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Solution Structure of a Type I Dockerin Domain, a Novel Prokaryotic, Extracellular Calcium-binding Domain
Abstract
The type I dockerin domain is responsible for incorporating its associated glycosyl hydrolase into the bacterial cellulosome, a multienzyme cellulolytic complex, via its interaction with a receptor domain (cohesin domain) of the cellulosomal scaffolding subunit. The highly conserved dockerin domain is characterized by two Ca(2+)-binding sites with sequence similarity to the EF-hand motif. Here, we present the three-dimensional solution structure of the 69 residue dockerin domain of Clostridium thermocellum cellobiohydrolase CelS. Torsion angle dynamics calculations utilizing a total of 728 NOE-derived distance constraints and 79 torsion angle restraints yielded an ensemble of 20 structures with an average backbone r.m.s.d. for residues 5 to 29 and 32 to 66 of 0.54 A from the mean structure. The structure consists of two Ca(2+)-binding loop-helix motifs connected by a linker; the E helices entering each loop of the classical EF-hand motif are absent from the dockerin domain. Each dockerin Ca(2+)-binding subdomain is stabilized by a cluster of buried hydrophobic side-chains. Structural comparisons reveal that, in its non-complexed state, the dockerin fold displays a dramatic departure from that of Ca(2+)-bound EF-hand domains. A putative cohesin-binding surface, comprised of conserved hydrophobic and basic residues, is proposed, providing new insight into cellulosome assembly.
... Crystallization of the dockerin domain is still unavailable to date; however, the structure of cellulosomal enzyme CelS from the C. thermocellum in solution has been solved [86]. The crystal structure of the cohesin-dockerin complex from C. ...
... Type I dockerins consist of about 70 residues and contain two 22-residue duplicated segments similar to the EF-hand type calcium-binding loop [20]. Solution structure of the C. thermocellum cellobiohydrolase CelS dockerin was described [86]. The The crystal structure for the cohesin-dockerin complex from C. thermocellum was reported which provided a clearer view of how a cohesin and a dockerin interacts [19]. ...
... Plasmid map and restriction map of pET23-coh6-pt-cbd engD ……………………………..86 ...
... A single structure of an isolated cellulosomal dockerin (Doc48S, PDB: 1DAV and 1DAQ) from the most abundance cellulosomal enzyme Cel48S of Clostridium thermocellum has been reported (Lytle et al., 2001), while several X-ray crystal structures of type-I dockerins in complex with their cognate type-I cohesin binding partners have been determined (Bras et al., 2012;Carvalho et al., 2003;Currie et al., 2012;Pinheiro et al., 2008). These latter structures revealed two nearly anti-parallel a-helices corresponding to a tandem duplicated amino acid sequence in the type-I dockerin modules, which forms two symmetric cohesin-binding sites resulting in a dual binding mode (Carvalho et al., 2003(Carvalho et al., , 2007Pinheiro et al., 2008). ...
... Owing to the observed discrepancies between the isolated Doc48S structure (Lytle et al., 2001) and the Xyn10B dockerin structure from the original type-I cohesin-dockerin complex (Carvalho et al., 2003), it had been proposed that the type-I dockerin module adopts a flexible conformation in solution, which undergoes a substantial conformational change upon binding to its cognate cohesin module. Nevertheless, Lytle et al. also reported the NMR backbone dynamics parameters, which showed that the average order parameter S 2 value for residues 5 to 29 and 32 to 66 corresponding to the structured regions is 0.81 ± 0.05, indicating that the structure is well ordered in solution (Lytle et al., 2001). ...
... Owing to the observed discrepancies between the isolated Doc48S structure (Lytle et al., 2001) and the Xyn10B dockerin structure from the original type-I cohesin-dockerin complex (Carvalho et al., 2003), it had been proposed that the type-I dockerin module adopts a flexible conformation in solution, which undergoes a substantial conformational change upon binding to its cognate cohesin module. Nevertheless, Lytle et al. also reported the NMR backbone dynamics parameters, which showed that the average order parameter S 2 value for residues 5 to 29 and 32 to 66 corresponding to the structured regions is 0.81 ± 0.05, indicating that the structure is well ordered in solution (Lytle et al., 2001). Inspection of these data associated with this structure revealed that 1 H and 15 N chemical shift assignments were incomplete ($88% and $85% of backbone and side chain, respectively) and that no 13 C chemical shift assignments were made. ...
... Both cohesin species exhibit an identical nine-stranded β-sandwich with jelly-roll topology. Later an NMR solution structure of the dockerin module [17] was reported, which revealed a novel symmetrically oriented duplicated 'Fhand motif', comprising a calcium-binding loop and adjacent α-helix. ...
... The internal 2-fold symmetry and distinctive sequence homology between the two anti-parallel 'F-hand' motifs [17,18] implies that both dockerin motifs could theoretically interact with the cohesin in an identical manner. Nevertheless, the crystal structure of the cohesin-dockerin heterodimer indicated that the interaction with cohesin is asymmetric. ...
... The initial crystal structures of individual type-I cohesin modules from the C. thermocellum and C. cellulolyticum scaffoldins [15,16] revealed the overall shape and structural characteristics of the molecule, but the structures failed to provide insight into their dockerin-binding sites. Later, the solution structure of the free C. thermocellum type-I dockerin module from cellobiohydrolase Cel48S [17] was solved, revealing two duplicated F-hand motifs, each consisting of a calcium-binding loop in a classical pentagonal bi-pyramidal sphere arrangement, followed by an α-helix. The calcium-binding loop comprises 12 residues, of which five of the side chains (aspartate and asparagine residues) serve to co-ordinate the calcium ion inside the loop. ...
Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module.
... aspartic acids and asparagines) are highly conserved. In this context, calcium dependence of functional dockerin was demonstrated experimentally (Yaron et al., 1995;Choi and Ljungdahl, 1996;Pagès et al., 1997;Lytle et al., 2001). Both duplicated segments were shown to be involved in cohesin binding (Lytle and Wu, 1998;Fierobe et al., 1999). ...
... The presence of the duplicated segment suggests that the final structure model of the dockerin exhibits twofold symmetry. The dockerin domain remained difficult to crystallize, but a solution structure from the C. thermocellum family 48 CelS cellulosomal enzyme was eventually described (Lytle et al., 2001). It revealed two Ca 2+ -binding loop-helix motif that indeed resemble the EF-hand motif with the lack of the E-helices as predicted previously (Pagès et al., 1997). ...
... In addition, Ca 2þ -binding sites in several bacterial extracytoplasmic proteins were found to resemble the EF-hands. These include, for example, the soluble lytic transglycosylase from Escherichia coli [16], the periplasmic galactose-binding protein from Salmonella typhimurium [17], Bacillus anthracis protective antigen [18] and the dockerin domain of the extracellular cellulase complex from Clostridium thermocellum [19]. While all these proteins deviate in one way or another from the typical EF-hands, they all share the conserved Ca 2þ -binding motif Dx(D/N)xDG within the central loop of the EFhand domain. ...
... Based on the discovery of calmodulin-like proteins in bacteria, it has been argued that the EF-hands had originated in the prokaryotic kingdom [11,14]. While this could still be correct, one has to consider that the Ca 2þ -binding loop is found in bacteria in a variety of diverse structural contexts : helix^loop^helix in calerythrin [13] and soluble lytic transglycosylase [16] , helix^loop^strand in the periplasmic galactose-binding protein from S. typhimurium [17], coil^loop^helix in the dockerin domain [19] and in B. anthracis protective antigen [18], and as the C-terminal loop in the Excalibur domain (Fig. 1). This suggests that evolution of the EF-hand-like domains might be much more complex than previously appreciated and these domains could be much more widespread than was previously recognized, involving various modi¢cations of the structural elements surrounding the Ca 2þ -binding loop. ...
Extracellular Ca(2+)-dependent nuclease YokF from Bacillus subtilis and several other surface-exposed proteins from diverse bacteria are encoded in the genomes in two paralogous forms that differ by a approximately 45 amino acid fragment, which comprises a novel conserved domain. Sequence analysis of this domain revealed a conserved DxDxDGxxCE motif, which is strikingly similar to the Ca(2+)-binding loop of the calmodulin-like EF-hand domains, suggesting an evolutionary relationship between them. Functions of many of the other proteins in which the novel domain, named Excalibur (extracellular calcium-binding region), is found, as well as a structural model of its conserved motif are consistent with the notion that the Excalibur domain binds calcium. This domain is but one more example of the diversity of structural contexts surrounding the EF-hand-like calcium-binding loop in bacteria. This loop is thus more widespread than hitherto recognized and the evolution of EF-hand-like domains is probably more complex than previously appreciated.
... Type I dockerins, which are well conserved among cellulosome-synthesizing bacteria, are composed of a pair of a 22-residue segment spaced by a linker of 8-18 residues. Each segment contains an EF-hand calcium-binding loop [32], as illustrated in Fig. 1A. The consensus logo sequence of the dockerins of R. cellulolyticum indicates a high level of conservation, in particular for the aspartate residues at positions 2, 6, 10, 13,38,42,46, and 49, involved in Ca 2+ coordination (Fig. 1A,B). ...
Cellulosomes are complex nanomachines produced by cellulolytic anaerobic bacteria such as Ruminiclostridium cellulolyticum (formerly known as Clostridium cellulolyticum). Cellulosomes are composed of a scaffoldin protein displaying several cohesin modules on which enzymatic components can bind to through their dockerin module. Although cellulosomes have been studied for decades, very little is known about the dynamics of complex assembly. We have investigated the ability of some dockerin‐bearing enzymes to chase the catalytic subunits already bound onto a miniscaffoldin displaying a single cohesin. The stability of the preassembled enzyme–scaffoldin complex appears to depend on the nature of the dockerin, and we have identified a key position in the dockerin sequence that is involved in the stability of the complex with the cohesin. Depending on the residue occupying this position, the dockerin can establish with the cohesin partner either a nearly irreversible or a reversible interaction, independently of the catalytic domain associated with the dockerin. Site‐directed mutagenesis of this residue can convert a dockerin able to form a highly stable complex with the miniscaffoldin into a reversible complex forming one and vice versa. We also show that refunctionalization can occur with natural purified cellulosomes. Altogether, our results shed light on the dynamics of cellulosomes, especially their capacity to be remodeled even after their assembly is ‘achieved’, suggesting an unforeseen adaptability of their enzymatic composition over time.
... The first structure of an isolated type-I dockerin module, determined by NMR spectroscopy, showed the two Fhand motifs arranged in an antiparallel conformation with amino acid residues from the a-helices of the two Fmotifs forming a modest hydrophobic core. As such, the dockerin module appeared to possess an apparent loosely packed overall planar topology that when compared to the dockerin structure in complex with its cognate cohesin module suggested it underwent a substantial cohesin-induced conformational change [15]. The structure of this type-I dockerin module was recently reassessed, again by NMR spectroscopy, where a larger number and more diverse set of distance restraints were included in the structure calculation [16 ]. ...
Cohesins and dockerins are complementary interacting protein modules that form stable and highly specific receptor-ligand complexes. They play a crucial role in the assembly of cellulose-degrading multi-enzyme complexes called cellulosomes and have potential applicability in several technology areas, including biomass conversion processes. Here, we describe several exceptional properties of cohesin-dockerin complexes, including their tenacious biochemical affinity, remarkably high mechanostability and a dual-binding mode of recognition that is contrary to the conventional lock-and-key model of receptor-ligand interactions. We focus on structural aspects of the dual mode of cohesin-dockerin binding, highlighting recent single-molecule analysis techniques for its explicit characterization.
... These orphan motifs have been proposed as EF-hand-like or pseudo-EF-hand ( Table 2). Examples of CaBP mentioned above include: the typical helix-loop-helix EF-hand structure seen in calerythrin [33,109] and calsymin [98], the longer 15 residue Ca 2+ -binding loop as in the E. coli lytic transglycosylase B Slt35 [110], the lacking of the first helix or lacking of the second helix as described in the C. thermocellum dockerin and the Sphingomonas sp alginate-binding protein, respectively [111,112], the extracellular Ca 2+ -binding region found in several bacteria, which has a shorter loop containing 10 residue motif DxDxDGxxCE has been called "Excalibur" by Ridgen et al. [28]. ...
... Thus, the LysGH15 CHAP domain represents an ''EF-hand-like'' protein. As in the protective antigen from Bacillus anthracis (PDB ID: 1ACC) [31] and the dockerin from Clostridium thermocellum (PDB ID: 1DAQ) [32], the calcium-binding site of the LysGH15 CHAP domain lacks the exiting helix, forming a ''loop-F'' pattern. Notably, the functions of these proteins available in the PDB that contain a calcium-binding site are completely unrelated to those of the LysGH15 CHAP domain. ...
The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.
... Some 332 of these proteins differ in the length of the Ca 2+ -binding loop, which may be shorter or longer 333 than 12 residues, while other proteins deviate in the secondary structure flanking the Ca 2+ -334 binding loop [28,29]. These orphan motifs have been proposed as EF-hand-like or pseudo-EF-335 hand ( lacking of the second helix as described in the C. thermocellum dockerin and the Sphingomonas 339 sp alginate-binding protein, respectively [111,112], the extracellular Ca 2+ -binding region found 340 in several bacteria, which has a shorter loop containing 10 residue motif DxDxDGxxCE has been 341 called "Excalibur" by Ridgen et al [28]. a total of 99 members [126]. ...
... CtDocI is the type I dockerin module from exo--1,3-galactanase from C. thermocellum. Others are PDB entries 1CLC (20), 1DAV_A (26), 1DAQ_A (26), and 1OHZ_B (4). ...
A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase-treated AGP.
... A spectral overlay of the apo-and holo-forms of ScaADoc showcased distinctive changes such as the downfield shift position of the 6th residue (Gly1638) in the first F-hand Ca 2+ -binding loop, which was a common spectral attribute for the DocS and type-II DocX [9,10,34]. Despite these shared tendencies with the type-I and -II dockerins, the solution behaviour of ScaADoc is distinguished from the type-II XDoc in the following: (a) the Ca 2+ -dependent conformational change did not accompany an increase in the molecular weight as reflected by the NMR linewidth, and (b) it can be expressed as a single protein module whereas the X-module was shown to stabilize the type-II Doc in the context of the XDoc modular pair. ...
Phylogenetic analysis of known dockerins in Ruminococcus flavefaciens revealed a novel subtype, type-Ill, in the scaffoldin proteins, ScaA, ScaB, ScaC and ScaE. In this study, we explored the Ca(2+)-binding properties of the type-Ill dockerin from the ScaA scaffoldin (ScaADoc) using a battery of structural and biophysical approaches using circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, and nuclear magnetic resonance spectroscopy. Despite the lack of a second canonical Ca(2+)-binding loop, the behaviour of ScaADoc is similar with respect to other dockerin protein modules in terms of its responsiveness to Ca(2+) and affinity for the cohesin from the ScaB scaffoldin. Our results highlight the robustness of dockerin modules and how their Ca(2+)-binding properties can be exploited in the construction of designer cellulosomes. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS: ScaB cohesin and ScaADocbind by isothermal titration calorimetry (View interaction) ScaB cohesin and ScaADocbind by molecular sieving (View interaction) ScaADoc and ScaB cohesinbind bybiophysical (View interaction) ScaB cohesinbinds to ScaADoc by enzyme linked immunosorbent assay (View interaction).
... When dockerins and cohesins are produced in their free form, they usually exhibit low expression levels, low solubilities, and a tendency to aggregate (Adams et al., 2005;Fierobe et al., 1999;Lytle et al., 2001). Therefore, we chose to fuse them to carrier proteins that are known for their good expression levels and solubility, when expressed in Escherichia coli (Barak et al., 2005). ...
The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) to non-catalytic scaffoldin units (carrying multiple cohesins) in cellulosome, a multienzyme plant cell-wall degrading complex. This interaction is species-specific, for example, the enzyme-borne dockerin from Clostridium thermocellum bacteria binds to scaffoldin cohesins from the same bacteria but not to cohesins from Clostridium cellulolyticum and vice versa. We studied the role of interface residues, contributing either to affinity or specificity, by mutating these residues on the cohesin counterpart from C. thermocellum. The high affinity of the cognate interactions makes it difficult to evaluate the effect of these mutations by common methods used for measuring protein-protein interactions, especially when subtle discrimination between the mutants is needed. We described in this article an approach based on indirect enzyme-linked immunosorbent assay (ELISA) that is able to detect differences in binding between the various cohesin mutants, whereas surface plasmon resonance and standard ELISA failed to distinguish between high-affinity interactions. To be able to calculate changes in energy of binding (ΔΔG) and dissociation constants (K(d) ) of mutants relative to wild type, a pre-equilibrium step was added to the standard indirect ELISA procedure. Thus, the cohesin-dockerin interaction under investigation occurs in solution rather than between soluble and immobilized proteins. Unbound dockerins are then detected through their interaction with immobilized cohesins. Because our method allows us to assess the effect of mutations on particularly tenacious protein-protein interactions much more accurately than do other prevalent methods used to measure binding affinity, we therefore suggest this approach as a method of choice for comparing relative binding in high-affinity interactions. Copyright © 2012 John Wiley & Sons, Ltd.
... The results showed that four suspected residues may serve as recognition codes for interaction with the cohesin domain (Mechaly et al., 2000;Mechaly et al., 2001;Pagè s et al., 1997). The three-dimensional solution structure of the 69residue dockerin domain of a Clostridium thermocellum cellulosomal cellulase subunit was recently determined (Lytle et al., 2001). As predicted earlier (Bayer et al., 1998;Lytle et al., 2000;Pagè s et al., 1997), the structure consists of two Ca 2+ -binding loop-helix motifs connected by a linker; the E helices entering each loop of the classical EF-hand motif are absent from the dockerin domain. ...
Cellulose and associated polysaccharides, such as xylans, comprise the major portion of the plant cell wall as structural polymers. As the plants evolved and distributed first in the seas and then on land, following their demise, the accumulated cellulosic materials had to be assimilated and returned to nature. Thus the cellulose-degrading bacteria have evolved to complement lignin-degrading microbial systems for the purpose of restoring the tremendous quantities of organic components of the plant cell wall to the environment for continued life cycles of carbon and energy on the global scale. This chapter is a sequel to a previous chapter of the same title from the second edition of this treatise (Coughlan MP, Mayer F (1992) The cellulose-decomposing bacteria and their enzyme systems. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer K-H (eds) The prokaryotes, vol I, 2nd edn. Springer, New York, pp 459-516.) and represents an update of our own subsequent chapter (Bayer EA, Shoham Y, Lamed R (2006) Cellulose-decomposing prokaryotes and their enzyme systems. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes, vol 2, 3rd edn. Springer, New York, pp 578-617.) which appeared in the third edition. Although the basic elements of the previous chapters are still essentially up to date, the field of the cellulose-decomposing bacteria has since advanced greatly, owing to two major factors: (1) the advent, progression, and increasing facility of genome- and metagenome-sequencing efforts and (2) the current initiatives to utilize plant-derived biomass for the production of biofuels as an alternative to fossil fuels for an energy source. © 2013 Springer-Verlag Berlin Heidelberg. All rights are reserved.
... When dockerins and cohesins are produced in their free form, they usually exhibit low expression levels, low solubilities, and a tendency to aggregate (Adams et al., 2005;Fierobe et al., 1999;Lytle et al., 2001). Therefore, we chose to fuse them to carrier proteins that are known for their good expression levels and solubility, when expressed in Escherichia coli (Barak et al., 2005). ...
The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions.
... Thus, a simple cellulosomal enzyme is composed of a catalytic module tethered to a small ($50 residues) dockerin through a linker ( Fig. 10.1). The picture of enzyme efficiency and specific protein-protein interaction in the cohesin-dockerin couple became clearer through numerous structures of isolated catalytic modules or scaffolding modules that were established by X-ray diffraction or NMR studies, followed by solution structures of a dockerin module (Lytle et al., 2001) and crystal structures of cohesin/dockerin complexes (Carvalho et al., 2003;Pinheiro et al., 2008). The latter structures revealed, for two different bacterial cellulosomes, a dual binding mode of the dockerin to its cohesin partner. ...
Small-angle X-ray scattering (SAXS) is an increasingly popular method to obtain low-resolution structures of complex macromolecules and their complexes in solution, in part due to recent technical and computational advances that make this method more and more accessible. However, to obtain unambiguous molecular interpretation from SAXS envelopes, the efficient use of and combination with additional structural methods are crucial. The multimodular character of cellulases and their assemblage in the cellulosome are ideally analyzed by such a combination of structural methods. Here, we describe how information from different sources can be combined with SAXS to determine the molecular organization and we depict the recent advancements and trends that are leading to a more comprehensive picture of the molecular architecture of these multimodular enzymes and their organization in macro-assemblages such as cellulosomes.
... 15,16 However, the major focus has been in obtaining atomic resolution structural information of the cellulosome components through a 'piecemeal' approach, whereby representative isolated modules of the scaffoldin, catalytic and cellsurface subunits have been the targets of structure determination by X-ray crystallography and NMR spectroscopy. 17 These include several C. thermocellum catalytic modules, [18][19][20][21][22][23][24] the CipA carbohydratebinding module, 25 a DocI module, 26 the second and seventh CohI modules, 27,28 and the SdbA CohII module. 29 Recent structures of the type-I 8,9 and type-II 30 Coh-Doc complexes have extended this structural knowledge toward the molecular determinants driving cellulosome assembly. ...
Cellulosomes are large, multienzyme, plant cell wall-degrading protein complexes found affixed to the surface of a variety of anaerobic microbes. The core of the cellulosome is a noncatalytic scaffoldin protein, which contains several type-I cohesin modules that bind type-I dockerin-containing enzymatic subunits, a cellulose-binding module, an X module, and a type-II dockerin that interacts with type-II cohesin-containing cell surface proteins. The unique arrangement of the enzymatic subunits in the cellulosome complex, made possible by the scaffoldin subunit, promotes enhanced substrate degradation relative to the enzymes free in solution. Despite representative high-resolution structures of all of the individual modules of the cellulosome, this mechanism of enzymatic synergy remains poorly understood. Consequently, a model of the entire cellulosome and a detailed picture of intermodular contacts will provide more detailed insight into cellulosome activity. Toward this goal, we have solved the structure of a multimodular heterodimeric complex from Clostridium thermocellum composed of the type-II cohesin module of the cell surface protein SdbA bound to a trimodular C-terminal fragment of the scaffoldin subunit CipA to a resolution of 1.95 A. The linker that connects the ninth type-I cohesin module and the X module has elevated temperature factors, reflecting an inherent flexibility within this region. Interestingly, a novel dimer interface was observed between CipA and a second, symmetry-related CipA molecule within the crystal structure, mediated by contacts between a type-I cohesin and an X module of a symmetry mate, resulting in two intertwined scaffoldins. Sedimentation velocity experiments confirmed that dimerization also occurs in solution. These observations support the intriguing possibility that individual cellulosomes can associate with one another via inter-scaffoldin interactions, which may play a role in the mechanism of action of the complex.
... In the case of the dockerin, due to the absence of structural restraints imposed by cohesin-dockerin interactions, the dockerin adopts a flexible conformation in solution after dissociation from its cohesin partner, consistent with inspection of the crystal structures. 7,11 Particularly notable is the coil connecting helix 1 and 2, which is locked in the complex, but highly flexible and disordered in the isolated form. The ordered-to-disordered transition is reflected in the difference in the RMSF values of the backbone atoms: <1 Å in the bound structure, but $2 Å in the free structure. ...
The organization and assembly of the cellulosome, an extracellular multienzyme complex produced by anaerobic bacteria, is mediated by the high-affinity interaction of cohesin domains from scaffolding proteins with dockerins of cellulosomal enzymes. We have performed molecular dynamics simulations and free energy calculations on both the wild type (WT) and D39N mutant of the C. thermocellum Type I cohesin-dockerin complex in aqueous solution. The D39N mutation has been experimentally demonstrated to disrupt cohesin-dockerin binding. The present MD simulations indicate that the substitution triggers significant protein flexibility and causes a major change of the hydrogen-bonding network in the recognition strips-the conserved loop regions previously proposed to be involved in binding-through electrostatic and salt-bridge interactions between beta-strands 3 and 5 of the cohesin and alpha-helix 3 of the dockerin. The mutation-induced subtle disturbance in the local hydrogen-bond network is accompanied by conformational rearrangements of the protein side chains and bound water molecules. Additional free energy perturbation calculations of the D39N mutation provide differences in the cohesin-dockerin binding energy, thus offering a direct, quantitative comparison with experiments. The underlying molecular mechanism of cohesin-dockerin complexation is further investigated through the free energy profile, that is, potential of mean force (PMF) calculations of WT cohesin-dockerin complex. The PMF shows a high-free energy barrier against the dissociation and reveals a stepwise pattern involving both the central beta-sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the beta-barrel structure.
... The initial 12 residues of these duplicated sequences bear striking similarity to the consensus sequence of the calcium-binding loop in the EF-hand motif (Chauvaux et al., 1990), where residues at the calciumcoordinating positions 1, 3, 5, 9, and 12 are highly conserved (usually Asp and Asn), as is the glycine residue at the hinge position 6 ( Fig. 1) (Giallo et al., 1983). This similarity is, in effect, restricted to the two calcium-binding loops and their respective exiting F helix, thereby suggesting an 'F-hand motif' (Pagès et al., 1997;Lytle et al., 2001), which would in part distinguish the dockerin module from the classical EF-hand motif (Rigden & Galperin, 2004). The type-III dockerin modules from R. flavefaciens cellulosomal components are much more divergent, particularly in their second segment, where, in some cases, the identity of the calcium-binding loop is not immediately recognizable (Rincon et al., 2005(Rincon et al., , 2007. ...
The high-affinity cohesin-dockerin interaction was originally discovered as modular components, which mediate the assembly of the various subunits of the multienzyme cellulosome complex that characterizes some cellulolytic bacteria. Until recently, the presence of cohesins and dockerins within a bacterial proteome was considered a definitive signature of a cellulosome-producing bacterium. Widespread genome sequencing has since revealed a wealth of putative cohesin- and dockerin-containing proteins in Bacteria, Archaea, and in primitive eukaryotes. The newly identified modules appear to serve diverse functions that are clearly distinct from the classical cellulosome archetype, and the vast majority of parent proteins are not predicted glycoside hydrolases. In most cases, only a few such genes have been identified in a given microorganism, which encode proteins containing but a single cohesin and/or dockerin. In some cases, one or the other module appears to be missing from a given species, and in other cases both modules occur within the same protein. This review provides a bioinformatics-based survey of the current status of cohesin- and dockerin-like sequences in species from the Bacteria, Archaea, and Eukarya. Surprisingly, many identified modules and their parent proteins are clearly unrelated to cellulosomes. The cellulosome paradigm may thus be the exception rather than the rule for bacterial, archaeal, and eukaryotic employment of cohesin and dockerin modules.
... The integrity of this consortium and its ability to degrade crystalline cellulose is dependent on the presence of calcium (5,6). Calcium binding is essential for the folding of dockerin domains that bind tightly to the cohesin domains of the scaffoldin protein anchoring cellulosomal proteins to it (7,8). Binding of Ca 2+ not only is confined to dockerin domains but also extends to other components such as the endoglucanase CelD, where calcium is believed to stabilize the active site conformation of the enzyme and enhance its activity (9,10). ...
Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.
... Before the discovery of bacterial CaBPs, comparison of the Ca 2þ -binding sites in various eukaryotic proteins resulted in delineation of a conserved helix-loop-helix structural motif, with Ca 2þ coordination provided by: side chains of three residues from the loop, a backbone atom from another loop residue, a water molecule (often coordinated by the side chain of another loop residue), and the side chain of an acidic (usually Glu) residue in the second helix (see Refs [2,3] for recent reviews). This EF-hand structure [4] is present in various families of diverse proteins but inter-family sequence conservation is sometimes low [2,3] structures -Escherichia coli soluble lytic-transglycosylase (Protein Data Bank entry 1qut) [6], the dockerin domain from Clostridium thermocellum (PDB: 1daq) [7] and periplasmic galactose-binding proteins from E. coli and Salmonella typhimurium (PDB: 1 gcg) [8] -share the conserved Ca 2þ -binding motif Dx(D/N)xDG with the central loop of the EF-hand domain. Although Michiels et al. [1] refer to all of them as 'EF-hands', there appears to be little similarity beyond this conserved motif. ...
Future research Although the above studies have identified key aspects of HCMV latency and reactivation, several questions still remain: monocytes have proven to be one viral reservoir, but are there other HCMV reservoirs, such as endothelial or bone marrow stromal cells? Are these cells infected latently or persistently, and if infected persistently, can virus released from them infect resident haematopoietic progenitor cells? These questions are difficult to address in the human system, and are best approached in relevant animal models, such as rodent and nonhuman primate systems.
Leucine Rich Repeat (LRR) domains, are present in hundreds of thousands of proteins across all kingdoms of life and are typically involved in protein-protein interactions and ligand recognition. LRR domains are classified into eight classes and when examined in three dimensions seven of them form curved solenoid-like super-helices, also described as toruses, with a beta sheet on the concave (inside) and stacked alpha-helices on the convex (outside) of the torus. Here we present an overview of the least characterized 8th class of LRR proteins, the TpLRR-like LRRs, named after the Treponema pallidum protein Tp0225. Proteins from the TpLRR class differ from the proteins in all other known LRR classes by having a flipped curvature, with the beta sheet on the convex side of the torus and irregular secondary structure instead of helices on the opposite, now concave site. TpLRR proteins also present highly divergent sequence pattern of individual repeats and can associate with specific types of additional domains. Several of the characterized proteins from this class, specifically the BspA-like proteins, were found in human bacterial and protozoan pathogens, playing an important role in the interactions between the pathogens and the host immune system. In this paper we surveyed all existing experimental structures and selected AlphaFold models of the best-known proteins containing this class of LRR repeats, analyzing the relation between the pattern of conserved residues, specific structural features and functions of these proteins.
TMEM16A mediates calcium-activated transmembrane flow of chloride ion and a variety of physiological functions. The binding of cytoplasmic calcium ions of TMEM16A and the consequent conformational changes of it are the key issues to explore the relationship between its structure and function. In recent years, researchers have explored this issue through electrophysiological experiment, structure resolving, molecular dynamic simulation and other methods. The structures of TMEM16 family members resolved by cryo-Electron microscopy (cryo-EM) and X-ray crystallization provide the primarily basis for the investigation of the molecular mechanism of TMEM16A. However, the binding and activation mechanism of calcium ions in TMEM16A are still unclear and controversial. This review discusses four Ca2+ sensing sites of TMEM16A and analyze activation properties of TMEM16A by them, which will help to understand the structure-function relationship of TMEM16A and throw light on the molecular design targeting TMEM16A channel.
Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca²⁺) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca²⁺-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer of gram-negative bacteria. Here, we report the crystal structure of the linker region and the C-terminal domain of OmpA from Capnocytophaga gingivalis, which comprises five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well ordered architecture organized around two tightly coordinated Ca²⁺ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca²⁺-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds that help to fix a conserved glycine residue at its proper position for coordinating Ca²⁺. Additionally, despite lacking inter-repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.
Biocatalysts showcase the upper limit obtainable for high-speed molecular processing and transformation. Efforts to engineer functionality in synthetic nanostructured materials are guided by the increasing knowledge of evolving architectures, which enable controlled molecular motion and precise molecular recognition. The cellulosome is a biological nanomachine, which, as a fundamental component of the plant-digestion machinery from bacterial cells, has a key potential role in the successful development of environmentally-friendly processes to produce biofuels and fine chemicals from the breakdown of biomass waste. Here, the progress toward so-called "designer cellulosomes", which provide an elegant alternative to enzyme cocktails for lignocellulose breakdown, is reviewed. Particular attention is paid to rational design via computational modeling coupled with nanoscale characterization and engineering tools. Remaining challenges and potential routes to industrial application are put forward.
Cellulosomes produced by certain anaerobic microbes are commonly assembled frommultiple subunits to macromolecular machines, and are extracellular protein complexes, which could organize and coordinate a variety of enzymic components to synergistically and effectively degrade lignocelluloses. Cellulosomes are the main forces of anaerobic hydrolytic celluloses, and play a vital role in breaking and using crystalline celluloses. The highly-efficient degradation on lignocelluloses of cellulosomes come from their self-assembled complex high-level structures, and the complex structures of the different anaerobic microorganisms have the astonishing diversity. The research advances on the diversity of assembly mode, structure, and the artificial design of cellulosomes were introduced.
Cellulosomes are highly elaborate multi-enzyme complexes of Carbohydrate Active enZYmes (CAZYmes) secreted by cellulolytic microorganisms, which very effectively degrade the most abundant polymers on Earth, cellulose and hemicelluloses. Cellulosome assembly requires that a non-catalytic dockerin module found in cellulosomal enzymes binds to one of the various cohesin domains located in a large molecular scaffold called Scaffoldin. A diversity of cohesin -dockerin binding specificities have been described, the combination of which may result in complex plant cell wall degrading systems, maximising the synergy between enzymes in order to improve catalytic efficiency. Structural studies have allowed the spatial flexibility inherent to the cellulosomal system to be determined. Recent progress achieved from the study of the fundamental cohesin and dockerin units involved in cellulosome assembly will be reviewed.
Cellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin-containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E, respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome-localized protease inhibitors in Clostridia.
Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of 17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.
utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology. Anaerobic bacteria harness a multiprotein complex, termed the "cellulosome," for efficient degradation of the plant cell wall. This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose "cohesin" domains interact with corresponding "dockerin" domains of the enzymes. Here we report the structure of the cohesin-dockerin complex from Clostridium thermocellum at 2.2-Angstrom resolution. The data show that the beta-sheet cohesin domain interacts predominantly with one of the helices of the dockerin. Whereas the structure of the cohesin remains essentially unchanged, the loop-helix-helix-loop-helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained. Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both "halves" of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of "polycellulosomes." The structure provides an explanation for the lack of cross-species recognition between cohesin-dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies.
Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides
into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The
core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules
of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude
structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering
has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the
solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments
comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A
cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal
fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural
and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.
Thermostable proteins have always been of significant interest in several scientific areas. In the last decade, the developments in the field of molecular and structural biology have offered important insights into the determinants of protein thermostability. Following an introductory section on thermostable proteins, extremophilic organisms and the determinants of protein thermostability, this chapter covers applications of NMR to the study of thermo-resistant proteins. Particular attention has been paid to papers dealing with structural comparisons between proteins from thermophilic micro-organisms and their corresponding mesophilic counterparts, the structure–function relationships of proteins from thermophilic sources and the factors involved in protein thermostability. The data reported, highlight the accepted assumption that there is not a single determinant of protein thermostability, rather an optimal occurrence of several factors.
Bacterial protein domains known as dockerins serve as anchoring or tethering modules for the assembly of large extracellular cellulase complexes called cellulosomes. Dockerins function through interactions with another class of protein-interaction module, the cohesins, and both domain families are currently divided into two subclasses (type I and type II) originally based on binding specificity. Dockerin sequences contain a pair of 22-residue motifs with homology to calcium-binding loops of the EF-hand family. When exposed to calcium, the dockerin adopts a novel fold distinct from EF-hand proteins that binds its cognate cohesin with high affinity. Conversely, in the absence of calcium, the dockerin domain is unfolded and does not bind cohesin. Thus, dockerin domains serve as calcium-regulated mediators of protein complex formation. Analysis of conserved sequence features in the context of the three-dimensional dockerin structure may identify elements that confer class and species specificity of dockerin–cohesin binding reactions.
Cellulosomes are multienzyme complexes responsible for efficient degradation of plant cell wall polysaccharides. The nonenzymatic scaffoldin subunit provides a platform for cellulolytic enzyme binding that enhances the overall activity of the bound enzymes. Understanding the unique quaternary structural elements responsible for the enzymatic synergy of the cellulosome is hindered by the large size and inherent flexibility of these multiprotein complexes. Herein, we have used x-ray crystallography and small angle x-ray scattering to structurally characterize a ternary protein complex from the Clostridium thermocellum cellulosome that comprises a C-terminal trimodular fragment of the CipA scaffoldin bound to the SdbA type II cohesin module and the type I dockerin module from the Cel9D glycoside hydrolase. This complex represents the largest fragment of the cellulosome solved by x-ray crystallography to date and reveals two rigid domains formed by the type I cohesin·dockerin complex and by the X module-type II cohesin·dockerin complex, which are separated by a 13-residue linker in an extended conformation. The type I dockerin modules of the four structural models found in the asymmetric unit are in an alternate orientation to that previously observed that provides further direct support for the dual mode of binding. Conserved intermolecular contacts between symmetry-related complexes were also observed and may play a role in higher order cellulosome structure. SAXS analysis of the ternary complex revealed that the 13-residue intermodular linker of the scaffoldin subunit is highly dynamic in solution. These studies provide fundamental insights into modular positioning, linker flexibility, and higher order organization of the cellulosome.
In this issue, van Bueren et al. (2011) take us on a journey from a functional link between the host glycogen-degrading properties of SpuA and Streptococcus pneumoniae virulence using cell-based studies, to revealing the structural basis for glycogen recognition and degradation by this S. pneumoniae virulence factor via complementary X-ray crystallography and small-angle X-ray (SAXS) studies.
Cellulosomes can be described as one of nature's most elaborate and highly efficient nanomachines. These cell bound multienzyme complexes orchestrate the deconstruction of cellulose and hemicellulose, two of the most abundant polymers on Earth, and thus play a major role in carbon turnover. Integration of cellulosomal components occurs via highly ordered protein:protein interactions between cohesins and dockerins, whose specificity allows the incorporation of cellulases and hemicellulases onto a molecular scaffold. Cellulosome assembly promotes the exploitation of enzyme synergism because of spatial proximity and enzyme-substrate targeting. Recent structural and functional studies have revealed how cohesin-dockerin interactions mediate both cellulosome assembly and cell-surface attachment, while retaining the spatial flexibility required to optimize the catalytic synergy within the enzyme complex. These emerging advances in our knowledge of cellulosome function are reviewed here.
The multimodular scaffoldin subunit CipA is the central component of the cellulosome, a multienzyme plant cell-wall-degrading complex, from Clostridium thermocellum. It captures secreted cellulases and hemicellulases and anchors the entire complex to the cell surface via high-affinity calcium-dependent interactions between cohesin and dockerin modules termed type I and type II interactions. The crystallization of a heterotrimeric complex comprising the type II cohesin module from the cell-surface protein SdbA, a trimodular C-terminal fragment of the scaffoldin CipA and the type I dockerin module from the CelD cellulase is reported. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 119.37, b = 186.31, c = 191.17 A. The crystals diffracted to 2.7 A resolution with four or eight molecules of the ternary protein complex in the asymmetric unit.
Abstract: To establish ionized calcium (Ca⁺²) and magnesium (Mg) requirements for growth (G) and cellulose degradation (CD) by cellulolytic rumen bacteria, F. succinogenes (FS) A3c and S85, R. albus (RA) 7 and 8, and R. flavefaciens (RF) B34b and C94 were incubated with cellobiose for G and cellulose for CD with varying Ca⁺² concentrations. Growth was measured at 600 nm and CD by substrate disappearance. Results were analyzed by a logistic function to obtain, max growth (MG), growth rate (GR) and lag time (LT) for G; extent of degradation (ED), degradation rate (DR) and LT for CD. Parameters were analyzed by ANOVA and linear and quadratic contrasts; maximum of the first derivative from quadratic functions were used to establish cation requirements. Ca⁺² concentrations affected G (MG, GR, LT) and CD (ED and LT) (P<0.05); strain*Ca⁺² concentrations was significant (P<0.05) for all G and CD parameters. MG Ca⁺² requirements are: FS-S85: 0.47mM, and RA-7: >0.64 mM (tendency); no estimation for A3c, B34b and C94. FS showed absolute Ca⁺² requirement for CD; Ca⁺² requirements for ED were: FS-Ac3: >0.36; RA-7: 0.28; RF-B34b and RF-C94: >0.64 mM; no estimation for S85. FS in NH3-free cellulose media confirmed their absolute Ca⁺² requirement for CD; requirements were: 0.42 and >0.64 mM for A3c and S85, respectively. A logistic function did not fit for RA-8; RG and RD were calculated by linear regression; positive effect of Ca⁺² concentration on RG (P<0.05), and no effect on RD (P>0.05) were found. No effects of Ca⁺² on MG and ED (P>0.05) for RA-8. RF showed an absolute Mg requirement for G; other Mg requirements for G are: FS-S85: 0.56; RA-7: 0.52; RA-8: 0.51; RF-B34b: 0.54 and RF-C94: >0.82 mM; for FS-A3c no estimation. For RF, NH3-free cellulose media was used to evaluate the effect of Ca⁺² and Mg on CD; no CD occurred when Mg was absent; no differences in ED for (plus-Ca⁺², plus-Mg) and (Ca⁺²-free, plus-Mg) (P>0.05), reflecting a Mg requirement for CD. Thus, Ca⁺² and Mg requirements differ among the strains within species, as well as by the substrate available. Title from first page of PDF file. Document formatted into pages; contains xvii, 163 p.; also includes graphics (some col.) Thesis (Ph. D.)--Ohio State University, 2005. Includes bibliographical references (p. 149-163). System requirements: World Wide Web browser and PDF viewer.
Ionized calcium (Ca(+2)) appears to be required by the 3 predominant species of rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. The present study evaluated the role of ionized calcium in growth and cellulose digestion. Maximum growth or rate and extent of digestion and lag time were the criteria used to evaluate Ca(+2) requirements. All cultures except F. succinogenes A3c grew when repeatedly transferred in a medium without added Ca(+2). As Ca(+2) concentration increased in cellobiose medium, the rate of growth increased and lag time decreased for F. succinogenes A3c, whereas F. succinogenes S85 exhibited increases in both maximum growth and rate of growth. No responses in any of the criteria were observed for the ruminococci in cellobiose medium. Both strains of F. succinogenes had an absolute requirement for Ca(+2) with cellulose as the only substrate. For strain A3c the requirement was 0.36 to 0.42 mM and for S85, >0.64 mM. Increases in extent of cellulose degradation occurred with all strains of ruminococci as Ca(+2) concentration increased; however, degradation in Ca(+2)-free medium was similar to that of F. succinogenes with Ca(+2). Although the ruminococci presumably have cellulosomes that require Ca(+2) in their structure, such was not evident in our studies. The function of Ca(+2) in cellulose degradation by F. succinogenes is unknown, but may be related to the secretion or activation of their cellulolytic enzymes. Based on reported concentrations of Ca(+2) in the rumen, it seems unlikely that an in vivo deficiency would occur for these bacteria.
The recycling of photosynthetically fixed carbon in plant cell walls is a key microbial process. In anaerobes, the degradation is carried out by a high molecular weight multifunctional complex termed the cellulosome. This consists of a number of independent enzyme components, each of which contains a conserved dockerin domain, which functions to bind the enzyme to a cohesin domain within the protein scaffoldin protein. Here we describe the first three-dimensional structure of a fungal dockerin, the N-terminal dockerin of Cel45A from the anaerobic fungus Piromyces equi. The structure contains a novel fold of 42 residues. The ligand binding site consists of residues Trp 35, Tyr 8 and Asp 23, which are conserved in all fungal dockerins. The binding site is on the opposite side of the N- and C-termini of the molecule, implying that tandem dockerin domains, seen in the majority of anaerobic fungal plant cell wall degrading enzymes, could present multiple simultaneous binding sites and, therefore, permit tailoring of binding to catalytic demands.
To locate the region involved in binding dockerin domains, 15 mutations were introduced across the surface of the seventh cohesin domain of the scaffolding protein CipA, which holds together the cellulosome of Clostridium thermocellum. Mutated residues were located on both faces of the nine-stranded beta-sandwich forming the cohesin domain and on the loops connecting beta-strands 4 and 5, 6 and 7, and 8 and 9. The loop region was previously proposed, on the basis of sequence comparisons, to form a contiguous "recognition strip". Individual mutants of four residues, D39, Y74, E86, and G89, formed no complexes detectable by nondenaturing gel electrophoresis after incubation with CelD664, a shortened form of endoglucanase CelD lacking the residues linking the catalytic domain with the dockerin domain. The four sensitive residues encompass a hydrophobic region on the 5-6-3-8 face of the molecule, which overlaps partially with the recognition strip and with a hydrophobic zone involved in the formation of cohesin-cohesin dimers. Isothermal titration calorimetry showed that single cohesin mutations affecting the binding of CelD664 had significant effects on the enthalpy or entropy of binding of wild-type CelD but much lesser effects on the association constant, owing to enthalpy-entropy compensation. However, the affinity for wild-type CelD of the triple mutant affecting D39, Y74, and E86 was reduced by 2 orders of magnitude, due to negative cooperativity between mutations affecting D39 + Y74 on one hand and E86 on the other hand.
Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosome-integrating protein CipA (type II) were constructed by swapping residues 10 and 11 of the first or the second duplicated segment between the two polypeptides. These residues have been proposed to determine the specificity of cohesin-dockerin interactions. The dockerin domain of CelD still bound to the seventh cohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Binding was no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. The dockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment was intact. None of the mutated dockerins displayed detectable binding to the noncognate cohesin domain. Isothermal titration calorimetry showed that binding of the CelD dockerin to CohCip7 occurred with a high affinity [K(a) = (2.6 +/- 0.5) x 10(9) M(-1)] and a 1:1 stoichiometry. The reaction was weakly exothermic (DeltaHdegrees = -2.22 +/- 0.2 kcal x mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal x mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal x mol(-1) x K(-1)). These values show that cohesin-dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segment reduced or enhanced, respectively, the hydrophobic character of the interaction. Due to partial enthalpy-entropy compensation, these mutations induced only small changes in binding affinity. However, the binding affinity was strongly decreased when both segments were mutated, indicating strong negative cooperativity between the two mutated sites.
The N-terminal type II cohesin from the cellulosomal ScaB subunit of Acetivibrio cellulolyticus was crystallized in two different crystal systems: orthorhombic (space group P2(1)2(1)2(1)), with unit-cell parameters a = 37.455, b = 55.780, c = 87.912 A, and trigonal (space group P3(1)21), with unit-cell parameters a = 55.088, b = 55.088, c = 112.553 A. The two crystals diffracted to 1.2 and 1.9 A, respectively. A selenomethionine derivative was also crystallized and exhibited trigonal symmetry (space group P3(1)21), with unit-cell parameters a = 55.281, b = 55.281, c = 112.449 A and a diffraction limit of 1.97 A. Initial phasing of the trigonal crystals was successfully performed by the SIRAS method using Cu Kalpha radiation with the selenomethionine derivative as a heavy-atom derivative. The structure of the orthorhombic crystal form was solved by molecular replacement using the coordinates of the trigonal form.
The utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology. Anaerobic bacteria harness a multiprotein complex, termed the "cellulosome," for efficient degradation of the plant cell wall. This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose "cohesin" domains interact with corresponding "dockerin" domains of the enzymes. Here we report the structure of the cohesin-dockerin complex from Clostridium thermocellum at 2.2-A resolution. The data show that the beta-sheet cohesin domain interacts predominantly with one of the helices of the dockerin. Whereas the structure of the cohesin remains essentially unchanged, the loop-helix-helix-loop-helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained. Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both "halves" of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of "polycellulosomes." The structure provides an explanation for the lack of cross-species recognition between cohesin-dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies.
A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an "X" domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature.
Natural protein complexes may provide the best templates for nanometer-scale technology and new biomaterials. The bacterial cellulosome is becoming a well-understood multi-protein complex found in cellulolytic microorganisms. The scaffoldin subunits of the bacterial cellulosome function to organize and position other protein subunits into the complex. The scaffoldins can also serve as an attachment device for harnessing the cellulosome onto the cell surface and/or for its targeting to substrate. Biochemical and molecular biological evidence have identified a receptor/adaptor type of protein domain pair, called "cohesin and dockerin," which is responsible for cellulosome self-assembly. The recognition between cohesin and dockerin is generally type and/or species specific. More than 80 cohesin and 100 dockerin sequences have been found, mostly from anaerobic bacteria. X-ray crystallography and NMR have been used to determine the three-dimensional structures of representative cohesin and dockerin domains, respectively. The cohesin peptide is about 140 amino acids in length and highly conserved in sequence and domain structure. The dockerin domain comprises about 70 amino acids and contains two 22 amino acid duplicated regions, each of which includes an "F-hand" modification of the EF-hand calcium-binding motif. Biochemical evidence and site-directed mutagenesis have confirmed that the two F-hand motifs are required for function and calcium dependence; at least two amino acids from each motif are critical for cohesin-dockerin recognition. In this report, we review the structure and function of the scaffoldin of the bacterial cellulosome and emphasize a detailed sequence analysis of the cohesin and dockerin domains. We also speculate about potential applications in nanoscience that may be based on cohesin-dockerin recognition.
A cellulose-binding, cellulase-containing factor, previously demonstrated to be responsible for the adherence of Clostridium thermocellum to cellulose, has been partly purified from cellulose-grown cells of this organism. The biochemical properties of the cell-associated factor were compared to those of the previously isolated extracellular factor, and a high degree of similarity was found in the properties and behavior of the two forms. Partial denaturation of the purified extracellular factor by treatment with sodium dodecyl sulfate at 25/sup 0/C, broke the complex into a reproducible pattern of smaller subcomplexes which were analyzed for their respective cellulolytic activities and corresponding subunit composition. The data indicate that a defined arrangement of endo- and exo-cellulases are organized in the parent complex. The term cellulosome is proposed for the cell-associated, cellulose-binding, multicellulase complex. 20 references, 8 figures, 2 tables.
The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.
The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced. When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed. The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da. The open reading frame is preceded by a Gram-positive-type ribosome binding site. A signal peptide sequence of 28 amino acids is present at its N terminus. The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues. There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids. These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex.
The cellulase complex from Clostridium cellulovorans has been purified and its subunit composition determined. The complex exhibits cellulase activity against crystalline cellulose as well as carboxymethylcellulase (CMCase) and cellobiohydrolase activities. Three major subunits are present with molecular masses of 170, 100, and 70 kDa. The 100-kDa subunit is the major CMCase, although at least four other, minor subunits show CMCase activity. The 170-kDa subunit has the highest affinity for cellulose, does not have detectable enzymatic activity, but is necessary for cellulase activity. Immunological studies indicate that the 170-kDa subunit is not required for binding of the catalytic subunits to cellulose and therefore does not function solely as an anchor protein. Thus this core subunit must have multiple functions. We propose a working hypothesis that the binding of the 170-kDa subunit converts the crystalline cellulose to a form that is capable of being hydrolyzed in a cooperative fashion by the associated catalytic subunits.
Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.
The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding. It appears that the CBF is not only responsible for the adherence of the cells to cellulose but also constitutes a major part of the cellulolytic apparatus of this organism.
The three-dimensional structure of calmodulin in the absence of Ca2+ has been determined by three- and four-dimensional heteronuclear NMR experiments, including ROE, isotope-filtering combined with reverse labelling, and measurement of more than 700 three-bond J-couplings. In analogy with the Ca(2+)-ligated state of this protein, it consists of two small globular domains separated by a flexible linker, with no stable, direct contacts between the two domains. In the absence of Ca2+, the four helices in each of the two globular domains form a highly twisted bundle, capped by a short anti-parallel beta-sheet. This arrangement is qualitatively similar to that observed in the crystal structure of the Ca(2+)-free N-terminal domain of troponin C.
The Clostridium thermocellum cellulosomal scaffolding protein, CipA, acts as an anchor on the cellulose surface for the various catalytic subunits of the cellulosome, a large extracellular cellulase complex. CipA contains nine repeated domains that serve as receptors for the cellulosomal catalytic subunits, each of which carries a conserved, duplicated ligand sequence (DS). Four representative CipA receptor domains with sequence dissimilarity were cloned and expressed in Escherichia coli. The interaction of these cloned receptor domains with the duplicated ligand sequence of CelS (expressed as a thioredoxin fusion protein, TRX-DSCelS), was studied by nondenaturing polyacrylamide gel electrophoresis. TRX-DSCelS formed a stable complex with each of the four receptor domains, indicating that CelS, the most abundant cellulosomal catalytic subunit, binds nonselectively to all of the CipA receptors. Conversely, the duplicated sequence of CipA (in the form of TRX-DSCipA), which is homologous to that of CelS, did not bind to any of the receptors under the experimental conditions.
The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa. SAA comprises an NH2-terminal leader peptide followed by three distinct regions. The NH2-terminal region is similar to the NH2-terminal repeats of C. thermocellum OlpB and ORF2p. The central region is rich in lysine and harbors a motif present in Streptococcus M proteins. The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins. The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA. Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined. Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.
This article reports the characterization of the Clostridium thermocellum SdbA protein thought to anchor the cellulosome to the bacterial cell surface. The NH2-terminal region of SdbA consists of a cohesin domain which specifically binds the dockerin domain of the cellulosomal scaffolding protein CipA. The COOH-terminal region consists of a triplicated segment, termed SLH repeats, which is present in the sequence of many bacterial cell surface polypeptides. The binding parameters of the interaction between the dockerin domain of CipA and the cohesin domain of SdbA were studied by using, as a probe, the chimeric polypeptide CelC-DSCipA, which carries the dockerin domain of CipA fused to endoglucanase CelC. In the presence of Ca2+, CelC-DSCipA bound to SdbA with an affinity constant of 1.26 x 10(7) M(-1). Binding of CelC-DSCipA to SdbA as a function of Ca2+ concentration was sigmoidal, corresponding to a Hill coefficient of 2 and an affinity constant for Ca2+ of 4 x 10(6) M(-2). This suggested the presence of two cooperatively bound Ca2+ ions in the cohesin-dockerin complex. Immunoblotting of C. thermocellum subcellular fractions and electron microscopy of immunocytochemically labeled cells indicated that SdbA is located on the cell surface and is a component of the cellulosome. Together, the data confirm that SdbA could mediate anchoring of the cellulosome to the surface of C. thermocellum cells by interacting with the dockerin domain of CipA.
The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca2+-bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction.
The Clostridium josui cipA and celD genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of C. josui CipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticum CipC, but C. josui CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of the C. josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.
The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279-2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013-1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 x 10(9) M-1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 x 10(8) M-1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly.
A novel cellulosomal scaffoldin gene, termed cipV, was identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of Clostridium thermocellum. The cipV gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted "recognition codes" differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of C. thermocellum and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain. Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from C. thermocellum as opposed to the known scaffoldins from the mesophilic clostridia. The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.
A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobeBacteroides cellulosolvens. The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437.
CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD),
and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins. In contrast to all other scaffoldins thus far described,
CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous
type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the
expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase
on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species,
would be considered to be type II. This is the first example of authentic type II cohesins that are confirmed components of
a cellulosomal scaffoldin subunit rather than a cell surface anchoring component. The results attest to the emerging diversity
of cellulosomes and their component sequences in nature.
The cross-species specificity of the cohesin–dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin–dockerin interaction seems to be species-specific. Based on intra- and cross-species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface. Proteins 29:517–527, 1997. © 1997 Wiley-Liss, Inc.
It is known that two proteins of the cellulosomal complex of Clostridium thermocellum (SL and Ss) together degrade crystalline cellulose. SL is a glycoprotein of 210000Da which enhances the binding to cellulose and the activity of Ss, an endoglucanase of 83000 Da. We have previously reported the cloning of a DNA fragment encoding the N-terminal end of the SL protein using antibodies raised against the native protein. A chromosomal walking approach using an EcoRI and a BamHI-Sau3A gene library allowed us to isolate the C-terminal end of the gene. Sequencing of both fragments revealed the existence of a leader peptide as has been found in cellulases of the same organism. This leader sequence is followed by a stretch of 14 amino acids that is identical to the N-terminal amino acid sequence of the native secreted protein. The open reading frame (ORF) of this gene encodes a protein of 196800 Da and is followed by a hairpin loop that could be involved in transcription termination. Within the open reading frame (ORF), we found nine internal repeated elements (IREs) of about 500 nucleotides each. Seven of these sequences displayed 98–100% homology and were located adjacent to each other within the structural gene without intervening regions. The remaining two, located on the N-terminal end of the gene, showed a significantly lower homology. Bearing in mind the inherent instability of reiterated regions, we confirmed the authenticity of our clones by Southern blot analysis using chromosomal C. thermocellum DNA and ruled out the possibility of rearrangements during the cloning and sequencing process. The sequenced gene is designated clpA and the encoded SL protein CipA.
Regulation of contraction in skeletal muscle occurs through calcium binding to the protein troponin C. The solution structures of the regulatory domain of apo and calcium-loaded troponin C have been determined by multinuclear, multidimensional nuclear magnetic resonance techniques. The structural transition in the regulatory domain of troponin C on calcium binding involves an opening of the structure through large changes in interhelical angles. This leads to the increased exposure of an extensive hydrophobic patch, an event that triggers skeletal muscle contraction.
The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517–527) we predicted that four amino acid residues of the ∼70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170–177. © 2000 Wiley-Liss, Inc.
A new program package, XEASY, was written for interactive computer support of the analysis of NMR spectra for three-dimensional structure determination of biological macromolecules. XEASY was developed for work with 2D, 3D and 4D NMR data sets. It includes all the functions performed by the precursor program EASY, which was designed for the analysis of 2D NMR spectra, i.e., peak picking and support of sequence-specific resonance assignments, cross-peak assignments, cross-peak integration and rate constant determination for dynamic processes. Since the program utilizes the X-window system and the Motif widget set, it is portable on a wide range of UNIX workstations. The design objective was to provide maximal computer support for the analysis of spectra, while providing the user with complete control over the final resonance assignments. Technically important features of XEASY are the use and flexible visual display of strips, i.e., two-dimensional spectral regions that contain the relevant parts of 3D or 4D NMR spectra, automated sorting routines to narrow down the selection of strips that need to be interactively considered in a particular assignment step, a protocol of resonance assignments that can be used for reliable bookkeeping, independent of the assignment strategy used, and capabilities for proper treatment of spectral folding and efficient transfer of resonance assignments between spectra of different types and different dimensionality, including projected, reduced-dimensionality triple-resonance experiments.
In the preceding paper it has been shown that the unique dynamic information on fast internal motions in an NMR relaxation experiment on macromolecules in solution is specified by a generalized order parameter, 8, and an effective correlation time, 7,. This paper deals with the extraction and interpretation of this information. The procedure used to obtain S2 and T, from experimental data by using a least-squares method and, in certain favorable circumstances, by using an analytical formula is described. A variety of experiments are then analyzed to yield information on the time scale and spatial restriction of internal motions of isoleucines in myoglobin, methionines in dihydrofolate reductase and myoglobin, a number of aliphatic residues in basic pancreatic trypsin inhibitor, and ethyl isocyanide bound to myoglobin, hemoglobin, and aliphatic side chains in three random-coil polymers. The numerical values of S2 and 7, can be readily interpreted within the framework of a variety of models. In this way, one can obtain the same physical picture of internal motions as that obtained by using complicated spectral densities to fit the data. The numerical value of the order parameter, unlike the effective correlation time T,, plays a crucial role in determining what models can be used to describe the experiment; models in which the order parameter cannot be reproduced are eliminated. Conversely, any model that can yield the correct value of S works.
MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20–40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.
A new approach to the interpretation of nuclear magnetic resonance relaxation experiments on macromolecules in solution is presented. This paper deals with the theoretical foundations and establishes the range of validity of this approach, and the accompanying paper demonstrates how a wide variety of experimental relaxation data can be successfully analyzed by using this approach. For both isotropic and anisotropic overall motion, it is shown that the unique imformation on fast internal motions contained in relaxation experiments can be completely specified by two model-independent quantities; (1) a generalized order parameter, S, which is a measure of the spatial restriction of the motion, and (2) an effective correlation time, T/sub e/, which is a measure of the rate of motion. A simple expression for the spectral density involving these two parameters is derived and is shown to be exact when the internal (but not overall) motions are in the extreme narrowing limit. The model-free approach (so called because S² and T/sub e/ have model-independent significance) consists of using the above spectral density to least-squares fit relaxation data by treating S² and T/sub e/ as adjustable parameters. The range of validity of this approach is illustrated by analyzing error-free relaxation data generated by using sophisticated dynamical models. Empirical rules are presented that allow one to estimate the of S² and T/sub e/ extracted by using the model-free approach by considering their numerical values, the resonance frequencies, and the parameters for the overall motion. For fast internal motions, it is unnecessary to use approaches based on complicated spectral densities derived within the framework of a model because all models that can give the correct value of S² work equally well.
Previous studies by Wishart et al. [Wishart, D. S., Sykes, B. D., & Richards, F. M. (1991) J. Mol. Biol. (in press)] have demonstrated that 1H NMR chemical shifts are strongly dependent on the character and nature of protein secondary structure. In particular, it has been found that the 1H NMR chemical shift of the alpha-CH proton of all 20 naturally occurring amino acids experiences an upfield shift (with respect to the random coil value) when in a helical configuration and a comparable downfield shift when in a beta-strand extended configuration. On the basis of these observations, a technique is described for rapidly and quantitatively determining the identity, extent, and location of secondary structural elements in proteins based on the simple inspection of the alpha-CH 1H resonance assignments. A number of examples are provided to demonstrate both the simplicity and the accuracy of the technique. This new method is found to be almost as accurate as the more traditional NOE-based methods of determining secondary structure and could prove to be particularly useful in light of the recent development of sequential assignment techniques which are now almost NOE-independent [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667]. We suggest that this new procedure should not necessarily be seen as a substitute to existing rigorous methods for secondary structure determination but, rather, should be viewed as a complement to these approaches.
The function of the non-catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross-reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I-labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit. 125I-labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.
The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.
Ribonuclease H is an endonuclease that hydrolyzes the RNA moiety of RNA-DNA duplex molecules. Escherichia coli ribonuclease H is involved in DNA replication, and retroviral ribonuclease H is essential for reverse transcription of the viral genome. To characterize the intramolecular dynamical properties of E. coli ribonuclease H, spin-lattice relaxation rate constants, spin-spin relaxation rate constants and steady state nuclear Overhauser effects for the 15N nuclear spins were measured by using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed by using a series of dynamical models in conjunction with a statistical model selection protocol. Ribonuclease H exhibits a complex array of dynamical features, most notably in the parallel beta-strands of the principal five-stranded beta-sheet, the coiled-coil helical interface, the active site, and the loop regions surrounding the active site. The dynamical properties are correlated with local structural environments of the 15N spins and suggest possible relationships to the functional properties of ribonuclease H. Results for E. coli ribonuclease H are compared to previously reported results for the human immunodeficiency virus type 1 ribonuclease H domain of reverse transcriptase.
Regulation of contraction in skeletal muscle occurs through calcium binding to the protein troponin C. The solution structures of the regulatory domain of apo and calcium-loaded troponin C have been determined by multinuclear, multidimensional nuclear magnetic resonance techniques. The structural transition in the regulatory domain of troponin C on calcium binding involves an opening of the structure through large changes in interhelical angles. This leads to the increased exposure of an extensive hydrophobic patch, an event that triggers skeletal muscle contraction.
The cellulases of many cellulolytic bacteria are organized into discrete multienzyme complexes, called cellulosomes. The multiple subunits of cellulosomes are composed of numerous functional domains, which interact with each other and with the cellulosic substrate. One of these subunits comprises a distinctive new class of noncatalytic scaffolding polypeptide, which selectively integrates the various cellulase and xylanase subunits into the cohesive complex. Intelligent application of cellulosome hybrids and chimeric constructs of cellulosomal domains should enable better use of cellulosic biomass and may offer a wide range of novel applications in research, medicine and industry.
The enzymatic subunits of the cellulosome of Clostridium thermocellum are integrated into the complex by a major non-catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single cellulose-binding domain (CBD) and nine subunit-binding domains, or cohesin domains. Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix. Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits. Calcium ions enhanced dramatically this binding. Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly.
The recombinant CelS (rCelS), the most abundant catalytic subunit of the Clostridium thermocellum cellulosome, displayed typical exoglucanase characteristics, including (i) a preference for amorphous or crystalline cellulose over carboxymethyl cellulose, (ii) an inability to reduce the viscosity of a carboxymethyl cellulose solution, and (iii) the production of few bound reducing ends on the solid substrate. The hydrolysis products from crystalline cellulose were cellobiose and cellotriose at a ratio of 5:1. The rCelS activity on amorphous cellulose was optimal at 70 degrees C and at pH 5 to 6. Its thermostability was increased by Ca2+. Sulfhydryl reagents had only a mild adverse effect on the rCelS activity. Cellotetraose was the smallest oligosaccharide substrate for rCelS, and the hydrolysis rate increased with the substrate chain length. Many of these properties were consistent with those of the cellulosome, indicating a key role for CelS.
Intracellular calcium plays an essential role in the transduction of most hormonal, neuronal, visual, and muscle stimuli. (Recent reviews include Putney, 1993; Berridge, 1993a,b; Tsunoda, 1993; Gnegy, 1993; Bachs et al. 1992; Hanson & Schulman, 1992; Villereal & Byron, 1992; Premack & Gardner, 1992; Means et al. 1991).
With a rapidly growing pool of known tertiary structures, the importance of protein structure comparison parallels that of sequence alignment. We have developed a novel algorithm (DALI) for optimal pairwise alignment of protein structures. The three-dimensional co-ordinates of each protein are used to calculate residue-residue (C alpha-C alpha) distance matrices. The distance matrices are first decomposed into elementary contact patterns, e.g. hexapeptide-hexapeptide submatrices. Then, similar contact patterns in the two matrices are paired and combined into larger consistent sets of pairs. A Monte Carlo procedure is used to optimize a similarity score defined in terms of equivalent intramolecular distances. Several alignments are optimized in parallel, leading to simultaneous detection of the best, second-best and so on solutions. The method allows sequence gaps of any length, reversal of chain direction and free topological connectivity of aligned segments. Sequential connectivity can be imposed as an option. The method is fully automatic and identifies structural resemblances and common structural cores accurately and sensitively, even in the presence of geometrical distortions. An all-against-all alignment of over 200 representative protein structures results in an objective classification of known three-dimensional folds in agreement with visual classifications. Unexpected topological similarities of biological interest have been detected, e.g. between the bacterial toxin colicin A and globins, and between the eukaryotic POU-specific DNA-binding domain and the bacterial lambda repressor.
Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.
The scaffoldin component of the cellulolytic bacterium Clostridium thermocellum is a non-hydrolytic protein which organizes the hydrolytic enzymes in a large complex, called the cellulosome. Scaffoldin comprises a series of functional domains, amongst which is a single cellulose-binding domain and nine cohesin domains which are responsible for integrating the individual enzymatic subunits into the complex. The cohesin domains are highly conserved in their primary amino acid sequences. These domains interact with a complementary domain, termed the dockerin domain, one of which is located on each enzymatic subunit. The cohesin-dockerin interaction is the crucial interaction for complex formation in the cellulosome. The determination of structural information about the cohesin domain will provide insights into cellulosome assembly and activity.
We have determined the three-dimensional crystal structure of one of the cohesin domains from C. thermocellum (cohesin 2) at 2.15 A resolution. The domain forms a nine-stranded beta sandwich with a jelly-roll topology, somewhat similar to the fold displayed by its neighboring cellulose-binding domain.
The compact nature of the cohesin structure and its lack of a defined binding pocket suggests that binding between the cohesin and dockerin domains is characterized by interactions between exposed surface residues. As the cohesin-dockerin interaction appears to be rather nonselective, the binding face would presumably be characterized by surface residues which exhibit both intraspecies conservation and interspecies dissimilarity. Within the same species, unconserved surface residues may reflect the position of a given cohesin domain within the scaffoldin subunit, its orientation and interactions with neighboring domains.
The new program DYANA (DYnamics Algorithm for Nmr Applications) for efficient calculation of three-dimensional protein and nucleic acid structures from distance constraints and torsion angle constraints collected by nuclear magnetic resonance (NMR) experiments performs simulated annealing by molecular dynamics in torsion angle space and uses a fast recursive algorithm to integrate the equations of motions. Torsion angle dynamics can be more efficient than molecular dynamics in Cartesian coordinate space because of the reduced number of degrees of freedom and the concomitant absence of high-frequency bond and angle vibrations, which allows for the use of longer time-steps and/or higher temperatures in the structure calculation. It also represents a significant advance over the variable target function method in torsion angle space with the REDAC strategy used by the predecessor program DIANA. DYANA computation times per accepted conformer in the "bundle" used to represent the NMR structure compare favorably with those of other presently available structure calculation algorithms, and are of the order of 160 seconds for a protein of 165 amino acid residues when using a DEC Alpha 8400 5/300 computer. Test calculations starting from conformers with random torsion angle values further showed that DYANA is capable of efficient calculation of high-quality protein structures with up to 400 amino acid residues, and of nucleic acid structures.
The quaternary organization of the cellulosome, a multi-enzymatic extracellular complex produced by cellulolytic bacteria, depends on specific interactions between dockerin domains, double EF-hand subunits carried by the catalytic components, and cohesin domains, individual receptor subunits linearly arranged within a non-catalytic scaffolding polypeptide. Cohesin-dockerin complexes with distinct specificities are also thought to mediate the attachment of cellulosomes to the cell membrane. We report here the crystal structure of a single cohesin domain from the scaffolding protein of Clostridium thermocellum. The cohesin domain folds into a nine-stranded beta-sandwich with an overall "jelly roll" topology, similar to that observed in bacterial cellulose-binding domains. Surface-exposed patches of conserved residues promote extensive intermolecular contacts in the crystal, and suggest a possible binding target for the EF-hand pair of the cognate dockerin domain. Comparative studies of cohesin domains indicate that, in spite of low sequence similarities and different functional roles, all cohesin domains share a common nine-stranded beta-barrel fold stabilized by a conserved hydrophobic core. The formation of stable cohesin-dockerin complexes requires the presence of Ca2+. However, the structure of the cohesin domain reported here reveals no obvious Ca2+-binding site, and previous experiments have failed to detect high affinity binding of Ca2+ to the unliganded dockerin domain of endoglucanase CelD. Based on structural and biochemical evidence, we propose a model of the cohesin-dockerin complex in which the dockerin domain requires complexation with its cohesin partner for protein stability and high-affinity Ca2+ binding.
Calcyclin is a member of the S100 subfamily of EF-hand Ca(2+)-binding proteins. This protein has implied roles in the regulation of cell growth and division, exhibits deregulated expression in association with cell transformation, and is found in high abundance in certain breast cancer cell lines. The novel homodimeric structural motif first identified for apo calcyclin raised the possibility that S100 proteins recognize their targets in a manner that is distinctly different from that of the prototypical EF-hand Ca2+ sensor, calmodulin. The NMR solution structure of Ca(2+)-bound calcyclin has been determined in order to identify Ca(2+)-induced structural changes and to obtain insights into the mechanism of Ca(2+)-triggered target protein recognition.
The three-dimensional structure of Ca(2+)-bound calcyclin was calculated with 1372 experimental constraints, and is represented by an ensemble of 20 structures that have a backbone root mean square deviation of 1.9 A for the eight helices. Ca(2+)-bound calcyclin has the same symmetric homodimeric fold as observed for the apo protein. The helical packing within the globular domains and the subunit interface also change little upon Ca2+ binding. A distinct homology was found between the Ca(2+)-bound states of the calcyclin subunit and the monomeric S100 protein calbindin D9k.
Only very modest Ca(2+)-induced changes are observed in the structure of calcyclin, in sharp contrast to the domain-opening that occurs in calmodulin and related Ca(2+)-sensor proteins. Thus, calcyclin, and by inference other members of the S100 family, must have a different mode for transducing Ca2+ signals and recognizing target proteins. This proposal raises significant questions concerning the purported roles of S100 proteins as Ca2+ sensors.
S100B is a homodimeric member of the EF-hand calcium-binding protein superfamily. The protein has been implicated in cellular processes such as cell differentiation and growth, plays a role in cytoskeletal structure and function, and may have a role in neuropathological diseases, such as Alzheimers. The effects of S100B are mediated via its interaction with target proteins. While several studies have suggested that this interaction is propagated through a calcium-induced conformational change, leading to the exposure of a hydrophobic region of S100B, the molecular details behind this structural alteration remain unclear.
The solution structure of calcium-saturated human S100B (Ca(2+)-S100B) has been determined by heteronuclear NMR spectroscopy. Ca(2+)-S100B forms a well defined globular structure comprising four EF-hand calcium-binding sites and an extensive hydrophobic dimer interface. A comparison of Ca(2+)-S100B with apo S100B and Ca(2+)-calbindin D9k indicates that while calcium-binding to S100B results in little change in the site I EF-hand, it induces a backbone reorientation of the N terminus of the site II EF-hand. This reorientation leads to a dramatic change in the position of helix III relative to the other helices.
The calcium-induced reorientation of calcium-binding site II results in the increased exposure of several hydrophobic residues in helix IV and the linker region. While following the general mechanism of calcium modulatory proteins, whereby a hydrophobic target site is exposed, the 'calcium switch' observed in S100B appears to be unique from that of other EF-hand proteins and may provide insights into target specificity among calcium modulatory proteins.
Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.
The growing database of three-dimensional structures of EF-hand calcium-binding proteins is revealing a previously unrecognized variability in the conformations and organizations of EF-hand binding motifs. The structures of twelve different EF-hand proteins for which coordinates are publicly available are discussed and related to their respective biological and biophysical properties. The classical picture of calcium sensors and calcium signal modulators is presented, along with variants on the basic theme and new structural paradigms.
Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.
Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop. To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy. Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure. This Ca(2+)-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and H(alpha) chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif.