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X-ray crystal structure of the complex of human leukocyte elastase (PMN Elastase) and the third domain of the turkey ovomucoid inhibitor
Abstract
Orthorhombic crystals diffracting beyond 1.7 A resolution, have been grown from the stoichiometric complex formed between human leukocyte elastase (HLE) and the third domain of turkey ovomucoid inhibitor (OMTKY3). The crystal and molecular structure has been determined with the multiple isomorphous replacement technique. The complex has been modeled using the known structure of OMTKY3 and partial sequence information for HLE, and has been refined. The current crystallographic R-value is 0.21 for reflections from 25 to 1.8 A resolution. HLE shows the characteristic polypeptide fold of trypsin-like serine proteinases and consists of 218 amino acid residues. However, several loop segments, mainly arranged around the substrate binding site, have unique conformations. The largest deviations from the other vertebrate proteinases of known spatial structure are around Cys168. The specificity pocket is constricted by Val190, Val216 and Asp226 to preferentially accommodate medium sized hydrophobic amino acids at P1. Seven residues of the OMTKY3-binding segment are in specific contact with HLE. This interaction and geometry around the reactive site are similar as observed in other complexes. It is the first serine proteinase glycoprotein analysed, having two sugar chains attached to Asn159 and to residue 109.
... In contrast, hPR3 exists as a tetramer in the form of a dimer of dimers in its only Tertiary structure of HLE and substrate-inhibitor recognition. (a) Overview of an HLE protomer in complex with a CQH ligand (black C atoms), with different colours for the N-and C-terminal domains and with labels for the consensus -strands; the side chains of the catalytic triad (Ser195/His57/ Asp102) are shown as well as a section of the proteinaceous inhibitor from the HLE structure with PDB code 1ppf (Bode et al., 1986; green C atoms; main chain as ribbon representation) in order to indicate the substrate-recognition sites. (b) Occupation of the S2 0 site and partial occupation of the S3 0 site of HLE, as indicated by the Tyr20 and Arg21 side chains of the proteinaceous inhibitor in the HLE structure with PDB code 1ppf, by the central Leu side chain and the terminal propyl group of CQH, respectively. ...
... Both domains contribute to the catalytic triad (His57, Asp102 and Ser195) and to an array of subsites that are critical for substrate binding and recognition (Fig. 3a). These subsites are structurally well characterized by three HLE structures in complex with proteinaceous inhibitors [PDB entries 1ppf (Bode et al., 1986), 2z7f (Koizumi et al., 2008) and 4nzl (Stapels et al., 2014)]. According to an established convention (Schechter & Berger, 1967) the specificity sites are designated S3, S2 and S1 at the N-terminal side of the substrate peptide bond to be hydrolysed and S1 0 , S2 0 and S3 0 at the C-terminal side (Fig. 3a). ...
... Even so, the absence of Asn72 glycosylation here is unsurprising since according to a recent comprehensive analysis (Loke et al., 2017) HLE glycosylation is subject to high macro-heterogeneity, with Asn72 only being glycosylated to 4%. Furthermore, the most frequent HLE isoform in granules of human leukocytes that prevails in commercially available batches and as has been established by HLE crystallography since the first structure determination (Bode et al., 1986) was found to be glycosylated only at Asn109 and Asn159 (Sinha et al., 1987). ...
Glycosylated human leukocyte elastase (HLE) was crystallized and structurally analysed in complex with a 1,3-thiazolidine-2,4-dione derivative that had been identified as an HLE inhibitor in preliminary studies. In contrast to previously described HLE structures with small-molecule inhibitors, in this structure the inhibitor does not bind to the S1 and S2 substrate-recognition sites; rather, this is the first HLE structure with a synthetic inhibitor in which the S2′ site is blocked that normally binds the second side chain at the C-terminal side of the scissile peptide bond in a substrate protein. The inhibitor also induces the formation of crystalline HLE dimers that block access to the active sites and that are also predicted to be stable in solution. Neither such HLE dimers nor the corresponding crystal packing have been observed in previous HLE crystal structures. This novel crystalline environment contributes to the observation that comparatively large parts of the N-glycan chains of HLE are defined by electron density. The final HLE structure contains the largest structurally defined carbohydrate trees among currently available HLE structures.
... In agreement with structural data, the catalytic activity of HNE increases with the length of the synthetic substrate, confirming the presence of an extended substrate-binding site [10]. The amino acid residues that probably form the specificity subsites of HNE have been discussed by Bode et al. [11]. The S1 pocket in each chymotrypsin-like serine proteases plays an important role in defining its substrate specificity. ...
... The S1 pocket in each chymotrypsin-like serine proteases plays an important role in defining its substrate specificity. The HNE S1 pocket is hemispherical and hydrophobic due to the presence of Val190, Phe192 Ala213, Val216, Phe228 and the disulfide bridge Cys191-Cys220 [11]. The S2 subsite of HNE is bordered by Phe215, Leu99 and the flat side of the imidazole ring of the catalytic triad His57, which is quite hydrophobic. ...
... secretory leukoprotease inhibitor (SLPI), inhibits HNE reversibly, with a K i of 4 3 10 211 M. Elafin, a 6 kDa protein from human keratinocytes or bronchial secretions is also a potent reversible inhibitor (K i 5 2 3 10 210 M) [5,18]. High-affinity reversible inhibitors of nonhuman origin include dog submandibulary gland inhibitor, eglin c from Hirudo medicinalis, guamerin from Hirudo nipponia [20], greglin from the locust Schistocerca gregaria [21], chemically engineered aprotinin (Trasylol), turkey ovomucoid, soybean trypsin inhibitor [5,11,22]. HNE is inhibited by non-specific irreversible serine proteinase inhibitors such as DFP and PMSF (from Sigma). More specific, commercially available irreversible inhibitors including MeOSuc-Ala-Ala-Pro-Val-CH 2 Cl (from Bachem) and DCI (from ICN) may be used to characterize HNE. ...
... When superimposed, the α-carbons of NE and CG only display a root mean square deviation (RMSD) of 0,9 Å (see chapter 1, figure 1.8). Since a RSMD value of zero means that structures are identical in conformation (Maiorov and Crippen 1994), it is apparent that the crystal structures of NE and CG are extremely similar (Bode et al. 1986b;Hof et al. 1996). Yet, NE cleaves virulence factors whereas CG does not. ...
... These residues were mainly located in the substrate-binding cleft formed by the ß-barrel domains of the enzymes. Interestingly, most of these amino acids were part of the previously described NE binding pockets (figure 3.10) (Bode et al. 1986b;Bode et al. 1989). ...
... Significant residues that had been suggested to influence the specificity of NE are shown in bold. This presentation is based on the crystallization of NE with the inhibitor TOM (Bode et al. 1986b) and adapted from (Bode et al. 1989) ...
Neutrophile Granulocyten wirken als einer der ersten Abwehrmechanismen gegen invasive Mikroorganismen im angeborenen Immunsystem von Mammalia. Aktiviert durch inflammatorische Signale verlassen diese Granulocyten das vaskuläre System und migrieren durch das Gewebe zum Infektionsherd. Dort binden sie die Mikroorganismen, phagozytieren und eliminieren diese schließlich mit hoher Effizienz. Humane Neutrophile Elastase (NE) ist Bestandteil der neutrophilen Granula und spielt eine entscheidende Rolle im Abbau von Virulenzfaktoren enteroinvasiver Bakterien, einschließlich der Shigella Virulenzfaktoren IpaB (invasion antigen plasmid B) und IcsA (intracellular spread A). NE gehört zu der Familie der Chymotrypsin-ähnlichen Serinproteasen, die sich durch Sequenz- und Strukurähnlichkeit auszeichnen, jedoch sehr unterschiedliche biologische Funktionen aufweisen. Cathepsin G (CG) ist wie NE eine Chymotrypsin-ähnliche Serinprotease und ebenfalls in neutrophilen Granula lokalisiert. Allerdings zeigt CG keine Aktivität gegenüber Virulenzfaktoren von Shigella. Obwohl die Kristallstrukturen von CG und NE fast identisch sind, konnten einzelne oder mehrere Aminosäuren in der Substratbindungsspalte identifiziert werden, die zwischen den beiden Enzymen differieren. Dies legte die Vermutung nahe, dass die Spezifität von NE gegenüber Virulenzfaktoren in diesen Unterschieden codiert sein könnte. Daher wurden diese Aminosäuren durch die analogen CG Aminosäuren oder durch Alanin ersetzt. Der Vergleich der funktionellen Eigenschaften der NE Mutanten mit wildtyp NE zeigte, dass die Aminosäuren an den Positionen 98 und 216-224 entscheidend für die Substratspezifität von NE sind. Die NE Mutanten N98A, 216-218 und 216-224 waren nicht mehr in der Lage, die Virulenzfaktoren IcsA und IpaB sowie das NE Peptidsubstrat abzubauen. Stattdessen haben diese Mutanten die Fähigkeit erlangt, das CG Peptidsubstrat abzubauen. Zusammenfassend konnten wir Aminosäuren in NE identifizieren, die sowohl die Spezifität von NE für das Peptidsubstrat als auch für die Virulenzfaktoren von Shigella flexneri determinieren.
... Afterwards, mature NSPs are mainly stored in granules. The active form of Human NE, PR3, and CG exhibits a three-dimensional structure containing two homologous β-barrels and a Cterminal α-helix (Bode et al., 1986;Hof et al., 1996). Each barrel contains six antiparallel β sheets that are connected through a linker segment, and the catalytic triad residues [Ser195, ...
... Asp102, His57 (chymotrypsin numbering)] are located at the junction of the two β-barrels, with the active site cleft running perpendicularly to the junction. Isolated mature Human NE, PR3, and CG from azurophilic granules have 218, 222, and 235 residues, respectively (Bode et al., 1986;Campanelli et al., 1990;Salvesen et al., 1987;Sinha et al., 1987) (Figure 4). Green letters indicate the conserved catalytic triad. ...
Homeobox (HOX) Gene kodieren für Transkriptionsfaktoren, welche essentiell für den korrekten Ablauf nicht nur der embryonalen Entwicklung sondern auch der Hämatopoiese sind. Eine Überexpression dieser Gene wird sowohl in akuter myeloischer sowie in akuter lymphatischer Leukämie (AML und ALL) beobachtet und ist mit einer schlechten Prognose verbunden. Bei den meisten Hox Genen führt eine Überexpression zur Leukämogenese, wobei der genaue Phänotyp je nach Hox Gen variiert. Hoxa9 zeigt hierbei die aggressivste Form der Krankheit. Bis jetzt ist jedoch wenig über den genauen Transformationsmechanismus, der letztendlich zur Leukämie führt, bekannt. Deshalb ist es wichtig, die grundlegenden regulatorischen Eigenschaften von Hoxa9 aufzuklären. Ein wichtiger Schritt hierfür ist es, die Bindeeigenschaften von Hoxa9 zu entschlüsseln und somit auch Signalwege zu analysieren, welche zur Entstehung von Leukämie beitragen. Es konnte bereits gezeigt werden, dass Hoxa9 während der Zelllyse degradiert wird. Durch weitere Versuche wurde dieses Phänomen mit den drei Proteasen Elastase (Elane), Proteinase 3 (Prtn3) und Cathepsin G (Ctsg) in Verbindung gebracht. Aufgrund dessen wurden, mit Hilfe von CRISPR-Cas9, Elane-Prtn3-Ctsg triple knockout Zellen generiert, in welchen Hoxa9 während der Zelllyse stabil ist. Diese Zellen konnten nun für eine ChIP-Analyse verwendet werden. Um Hoxa9-kontrollierte Elemente (Enhancer und Promotor) in murinen Hoxa9-transformierten myeloiden Zellen zu identifizieren, wurden Immunopräzipitationen von H3K27ac und H3K4me sowohl in „Hoxa9-on“ als auch in „Hoxa9-off“ Zellen durchgeführt. Außerdem wurden die Veränderungen nascenter RNA-Synthese, reguliert durch Hoxa9, in einem induzierbarem System analysiert. Es konnte gezeigt werden, dass Hoxa9 einen positiven feedback loop generiert, welcher die Expression von Hox A Genen steuert. Weiterhin konnte gezeigt werden, dass Hoxa9 die Expression bestimmter Onkogene, wie Myc oder Myb, induziert, sowie direkten Einfluss auf Regulatoren des Zellzyklus (Cdk6, CyclinD1) und auf Telomerase hat. Diese Arbeit liefert neue Erkenntnisse über die Hoxa9-induzierte Transformation und Leukämogenese.
... It can degrade a wide range of substrates including cartilage proteoglycan, fibronectin, laminin, coUagens (type I, II, and IV) and bacterial ceU waU proteins. It can also degrade plasma factors like fibrinogen, fibrin, and anti-thrombin IQ (Keiser et al., 1976;Thome et al., 1976;McDonald and Kelley, 1980;Mainardi et al., 1980;CampbeU et al., 1982;Bode et al., 1986;Pipoly and Crouch, 1987). ...
... In humans GE may be involved in the activation of the BPI protein by proteolytic cleavage ). hGE has been thought to play a role in the digestion of bacterial degradation products (Bode et al., 1986) and to facilitate the penetration of other neutrophil 42 antimicrobial molecules by proteolysis of bacterial cell wall components (Blondin and Janoff, 1976). hGE is capable of activating latent gelatinase, a component of tertiary granules in neutrophils, following neutrophil treatment with fMLP in the presence of c\toc!klasin B (Vissers and Winterboum, 1988). ...
Neutrophils form a major proportion of the phagocytic population in the body canying out the first line of defence against bacterial and fungal infections. The ingested microbes are killed by a combination of reactive oxygen radicals and antimicrobial proteases in the phagocytic vacuole. Discharge of reactive oxygen radicals and proteases by activated phagocytes into the extracellular space is thought to play a major role in inflammatory and other immunopathological reactions (e.g. emphysema, rheumatoid arthritis, inflammatory bowel disease). To determine the physiologic and pathologic function of granulocyte elastase (GE), one of the major proteases in neutrophils. we have cloned and inactivated the gene encoding GE by homologous recombination in mouse embryonic stem cells. Correctly targeted clones were introduced into blastocysts for the generation of chimeric mice used to establish a GE-deficient mouse line The GE-deficient mice were shown by Northern blot analysis to have no detectable GE mRNA Western blot analysis further showed absence of GE protein The GE-deficient mice appear to be phenotypically normal under the conditions encountered in the animal house facilities. Neutrophil numbers and neutrophil migration into the peritoneum following a sterile inflammatory stimulus are normal as is neutrophil degranulation. GE-deficient mice showed no increase in mortality rate when challenged with Staphylococcus aureus (S. aureus), a gram positive bacterial pathogen. Tissue clearance of the S. aureus infection by wild type and mutant mice was comparable with both types of mice developing acute, but not fatal, progressive pyelonephritis five days post-infection. Concomitant with GE inactivation, the P1 bacteriophage cre recombinase gene was introduced into the mouse genome 5' of GE so that its start of translation site directly replaced that of GE. The aim was to establish a granulocyte specific knockout program based on the Cre-loxp site-specific recombination process. Cre recombinase activity was shown to be prominent in granulocyte fractions and at much reduced levels in other tissues examined, probably resulting from contaminating granulocytes.
... Selection of Contact Positions. X-ray crystallography of complexes of OMTKY3, of many of its variants, and of some PSTI variants with SGPB, CHYM and HLE showed that a consensus set of 12 contact positions in the inhibitors is in contact with the enzymes (29)(30)(31)(32)(33)(34) (Fig. 2). When sequences of ovomucoid third domains from 153 species are compared, the seven most variable positions in the 51 residue domains all lie in the consensus contact residue set (24). ...
... The side chains of these two residues are hydrogenbonded to each other in the free inhibitor (37). The hydrogen bond shortens on complex formation with SGPB (29) and CHYM (30) but not with HLE (31). The same shortening is seen when the structure of variant 3 of PSTI (38) (this variant has P 2 Thr-17 and P 1 Ј Glu-19) is compared in free inhibitor and in complex with chymotrypsinogen (34). ...
An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed , and an evolutionary problem was solved.
... For easy identification of residues of HNE, the commonly used chymotrypsin numbering [60] scheme is used throughout this manuscript. scHNE undergoes self-cleavage between A188 and G189 to form a tcHNE with altered activity profile [48]. ...
Background:
Human neutrophil elastase (HNE) is a key driver of systemic and cardiopulmonary inflammation. Recent studies have established the existence of a pathologically active auto-processed form of HNE with reduced binding affinity against small molecule inhibitors.
Method:
AutoDock Vina v1.2.0 and Cresset Forge v10 software were used to develop a 3D-QSAR model for a series of 47 DHPI inhibitors. Molecular Dynamics (MD) simulations were carried out using AMBER v18 to study the structure and dynamics of sc (single-chain HNE) and tcHNE (two-chain HNE). MMPBSA binding free energies of the previously reported clinical candidate BAY 85-8501 and the highly active BAY-8040 were calculated with sc and tcHNE.
Results:
The DHPI inhibitors occupy the S1 and S2 subsites of scHNE. The robust 3D-QSAR model showed acceptable predictive and descriptive capability with regression coefficient of r2 = 0.995 and cross-validation regression coefficient q2 = 0.579 for the training set. The key descriptors of shape, hydrophobics and electrostatics were mapped to the inhibitory activity. In auto-processed tcHNE, the S1 subsite undergoes widening and disruption. All the DHPI inhibitors docked with the broadened S1'-S2' subsites of tcHNE with lower AutoDock binding affinities. The MMPBSA binding free energy of BAY-8040 with tcHNE reduced in comparison with scHNE while the clinical candidate BAY 85-8501 dissociated during MD. Thus, BAY-8040 may have lower inhibitory activity against tcHNE whereas the clinical candidate BAY 85-8501 is likely to be inactive.
Conclusion:
SAR insights gained from this study will aid the future development of inhibitors active against both forms of HNE.
... Thereby, NSPs have pro-and anti-inflammatory activities, shaping the immune response at the site of inflammation. The NSPs, NE, PR3 and CTSG share a common three dimensional structure consisting of a C-terminal α-helix and two homologous β-barrels (Bode et al. 1986). The active center comprises the catalytic triad (histidine, serine and aspartate) (Korkmaz et al. 2010). ...
Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterized by a severe inflammation and infiltration of neutrophiles which secrete neutrophil elastase (NE), which leads to lung parenchyma destruction and disease progression. In CF, disease severity has been linked with airway dysbiosis. As the relationship of dysbiosis, respiratory tract inflammation and impaired lung function is not fully understood this study aimed to assess this interplay. In an initial cross-sectional study, the inflammatory profile of spontaneous sputum samples of CF patients was analyzed. Cytokines were measured by Cytometric Bead Array, anti-proteases levels by enzyme-linked immunosorbent assay and free NE activity via Förster resonance energy transfer (FRET) probe NEmo-1 in the supernatant fraction. The interplay of those parameters together with lung function were evaluated in a multiparameter analysis. The increase in the inflammatory markers NE, IL-1β, IL-8 and TNF-α correlated negatively with the lung function parameter forced expiratory volume in one second percent (FEV1 %) predicted and α-diversity of the airway microbiome. Based on the microbiome composition, patients were grouped in 7 clusters. One cluster had a diverse microbiome, which was rich in species of the oropharynx-like flora and had a high evenness. These patients had the highest FEV1 % predicted and the lowest inflammatory load. In contrast, the dominance of a particular pathogen in other clusters was associated with higher levels of inflammation and lower lung function; this was most pronounced in the cluster dominated by Pseudomonas aeruginosa. The time-resolved interdependencies of the parameters was evaluated in a longitudinal follow-up study. Repeated sampling over a year revealed a strong decrease of the microbiome diversity while inflammatory markers were rather volatile. However, during a 3-year study, inflammatory markers continuously increased, and FEV1 % predicted declined. Moreover, the patients’ initial condition and disease stage had an important effect on the disease progression. Thus, the combined investigation of microbial clusters, inflammation parameters and lung function may provide information about disease severity and allow a more directed therapeutic treatment. Previous studies considered only free NE activity, though recent evidence suggests importance in pathogenesis of its membrane associated fraction. As a rapid and sensitive quantification method to measure membrane bound NE is missing, a flow cytometric approach based on the FRET probe NEmo-2E was established. With the new method it was shown that, a saturation of the membrane NE was detected in CF and COPD, whereas free NE activity was substantially elevated in CF sputum samples. This indicates that NE first associates to the membrane before excess NE is detectable in the supernatant fraction. Thus, membrane associated NE activity could be a potential early inflammation marker for both, COPD and CF.
... hPR3 and hNE share an analogous catalytic mechanism 16 and the elucidation of the crystal structure of hPR3 has confirmed that both enzymes display a common fold. 17,18 Despite hPR3 and hNE preferentially accommodate small hydrophobic residues in the S1 pocket, their different substrate specificities have been exploited to develop synthetic inhibitors and FRET peptidyl substrates of human PR3. [19][20][21][22] Although hPR3 is considered as a relevant therapeutic target, its precise contribution to some diseases remains still debated. ...
... Apart from the defensive role of NSPs, recent publications indicate a role in different disease conditions since their overexpression can damage host tissues [30e33]. NSPs are therefore associated with various inflammatory diseases like chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome, rheumatoid arthritis [31] and non-infectious inflammatory diseases like arthritis, bullous pemphigoid and glomerulonephritis [33,34]. In order to control the activity of these proteases a number of physiological and non-physiological inhibitors for NSPs regulation has been identified and reported [35e57]. ...
Neutrophils synthesize four immune associated serine proteases: Cathepsin G (CTSG), Elastase (ELANE), Proteinase 3 (PRTN3) and Neutrophil Serine Protease 4 (NSP4). While previously considered to be immune modulators, overexpression of neutrophil serine proteases correlates with various disease conditions. Therefore, identifying novel small molecules that can potentially control or inhibit the proteolytic activity of these proteases is crucial to revert or temper the aggravated disease phenotype. To the best of our knowledge, although there is limited data for inhibitors of other neutrophil protease members, there is no previous clinical study of a synthetic small molecule inhibitor targeting NSP4. In this study, an integrated molecular modeling algorithm was performed within a virtual drug repurposing study to identify novel inhibitors for NSP4, using clinically approved and investigation drugs library (∼8000 compounds). Based on our rigorous filtration, we found that following molecules Becatecarin, Iogulamide, Delprostenate and Iralukast are predicted to block the activity of NSP4 by interacting with core catalytic residues. The selected ligands were energetically more favorable compared to the reference molecule. The result of this study identifies promising molecules as potential lead candidates.
... 30 HNE preferentially cleaves on the C-terminus of small hydrophobic residues such as Gly, Ala, and Val as the substrate specicity is based on the characteristics of its S1 pocket, where it was shown to be hydrophobic and hemispherical. 31 The 3D structures of HNE and porcine pancreatic elastase were complexed with various inhibitors, providing useful information regarding the molecular determinant of selectivity and potency, which could aid in structure-based drug design of new analogues with improved overall proles. [32][33][34][35][36] The imbalance between neutrophil elastase (NE) and its endogenous inhibitors (e.g., elan and a1-antitrypsin) are features of many respiratory diseases characterized by exaggerated inammatory responses such as pneumonia, cystic brosis (CF), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and acute lung injury (ALI). ...
Covering: up to April 2020
Proteases are involved in the regulation of many physiological processes. Their overexpression and dysregulated activity are linked to diseases such as hypertension, diabetes, viral infections, blood clotting disorders, respiratory diseases, and cancer. Therefore, they represent an important class of therapeutic targets. Several protease inhibitors have reached the market and >60% of them are directly related to natural products, even when excluding synthetic natural product mimics. Historically, natural products have been a valuable and validated source of therapeutic agents, as over half of the marketed drugs across targets and diseases are inspired by natural product structures. In the past two decades the number of new protease inhibitors discovered from nature has sharply increased. Additionally, the availability of 3D structural information for proteases has permitted structure-based design and accelerated the synthesis of optimized lead structures with improved potency and selectivity profiles, resulting in some of the most-potent-in-class inhibitors. These discoveries were oftentimes maximized by in-depth biological assessments of lead inhibitors, linking them to a relevant disease state. This review will discuss some of the current and emerging drug targets and their involvement in various disease processes, highlighting selected success stories behind several FDA-approved protease inhibitors that have natural products scaffolds as well as recent selected pharmacologically well-characterized inhibitors derived from marine or terrestrial sources.
... In this manuscript, I used crystal structures of several serine proteases to illustrate the structure/function and thus specificity relationship. Protein Data Bank codes are listed in Elastase-like proteases have a hemispherical and hydrophobic S1 pocket because of the presence of Val190, Ala213, Val216, Ala227, Phe228 (Bode et al., 1986). As an illustration, elastases (NE & PE) and proteinase 3 prefer small alkyl side chains (such as alanine or valine) in P1 since their pocket is shallow. ...
This work focuses on substrate-based probes for proteolysis monitoring by Electron Paramagnetic Resonance spectroscopy (EPR) and for in vivo imaging by Overhauser-enhanced Magnetic Resonance (OMRI). More precisely, this work investigates for the first time a family of MRI agents named “line-shifting nitroxide” specific for proteolytic activities. Proteolytic action results in a shift of 5 G in EPR hyperfine coupling constants allowing individual quantification of substrate and product species by EPR and selective excitation by OMRI. Three substrates were worked out, showing enzymatic specificity for neutrophil elastase (MeO-Suc-Ala-Ala-Pro-Val-Nitroxide & Suc-Ala-Ala-Pro-Val-Nitroxide), and for Chymotrypsin/Cathepsin G (Suc-Ala-Ala-Pro-Phe-Nitroxide). Enzymatic constants were remarkably good with globally Km = 28 ± 25 µM and kcat = 19 ± 3 s-1. Ex vivo, the use of NE substrates in OMRI revealed a high contrast in bronchoalveolar lavages of mice under inflammatory stimulus. MRI signal enhancements correlate with the severity of inflammation. Irradiation at the RPE frequency of 5425.6 MHz provided access to the bio-distribution of substrates in vivo and could thus serve as a diagnostic tool. The medium-term perspectives of this work are based on the development of OMRI with very low magnetic fields for human application
... The S1 pocket of the active site is known to be hydrophobic due to the presence of Val190, Phe192, Ala213, Val216, Phe228, and a disulfide bridge at Cys191-Cys220. Therefore, small hydrophobic residues are needed in the P1 site of the substrate such as Val, Cys, Ala, Met, or Ile in order to achieve an enzymatic activity [9,10]. The S2 pocket of the enzyme contains Phe215 and Leu99 and the flat side of the imidazole ring of the catalytic triad His57, which give this site a hydrophobic nature. ...
Human neutrophil elastase is a proteolytic enzyme secreted by neutrophil during inflammation and infection in lung environment. It has an important anti-bacterial
activity through different pathways. However, it also has an important proteolytic action against lung’s extracellular matrix which lead to different disease related
to acute lung injuries. secretory leukocyte proteinase inhibitor (SLPI) is a naturally produced protein with an elastase inhibition main role. Different lung diseases
were linked to the imbalance of elastase/SLPI ratio in lung environment. In addition, SLPI has an important antibacterial activity. In this review, a new strategy was
suggested to use elastase and SLPI as therapeutic agents. Using encapsulation technology, the entrapment of these molecules inside albumin nanoparticles could
produce a great opportunity in tissue degradation protection and non-antibiotic related bacterial killing.
... This could, in principle, elaborate quite complex Fig. 20. Stereodrawing of the complex between human leucocyte elastase (thin lines) and turkey ovomucoid inhibitor (thick lines) ( Bode et al., 1986). structures within the aqueous channel (Lodish, 1988), but available evidence at low resolution for gap junction proteins ( Milks et al., 1988) indicates in this specific case a simple hexameric arrangement of membrane-spanning amphiphilic a-helices, whose polar sides face the aqueous channel. ...
Aspects of intramolecular light energy and electron transfer will be discussed for three protein‐‐cofactor complexes, whose three‐dimensional structures have been elucidated by X‐ray crystallography: components of light‐harvesting cyanobacterial phycobilisomes; the purple bacterial reaction centre; and the blue multi‐copper oxidases. A wealth of functional data is available for these systems which allows specific correlations between structure and function and general conclusions about light energy and electron transfer in biological materials to be made.
... NSPs display a 3D structure consisting of two homologous β-barrel domains and a C-terminal α-helix (Bode et al., 1986;Fujinaga, Chernaia, Halenbeck, Koths, & James, 1996;Hof et al., 1996). The highly conserved residues of the catalytic triad (His57, Asp102, Ser195; chymotrypsin numbering) are located at the interface between the two β-barrels. ...
Cathepsin C (CatC) is a highly conserved tetrameric lysosomal cysteine dipeptidyl aminopeptidase. The best characterized physiological function of CatC is the activation of pro-inflammatory granule-associated serine proteases. These proteases are synthesized as inactive zymogens containing an N-terminal pro-dipeptide, which maintains the zymogen in its inactive conformation and prevents premature activation, which is potentially toxic to the cell. The activation of serine protease zymogens occurs through cleavage of the N-terminal dipeptide by CatC during cell maturation in the bone marrow. In vivo data suggest that pharmacological inhibition of pro-inflammatory serine proteases would suppress or attenuate deleterious effects of inflammatory/auto-immune disorders mediated by these proteases. The pathological deficiency in CatC is associated with Papillon-Lefèvre syndrome. The patients however do not present marked immunodeficiency despite the absence of active serine proteases in immune defense cells. Hence, the transitory pharmacological blockade of CatC activity in the precursor cells of the bone marrow may represent an attractive therapeutic strategy to regulate activity of serine proteases in inflammatory and immunologic conditions. A variety of CatC inhibitors have been developed both by pharmaceutical companies and academic investigators, some of which are currently being employed and evaluated in preclinical/clinical trials.
... Another critical residue that makes the specificities of humPR3 and humNE different is that at position 217 in the vicinity of the S4 subsite where an Ile in humPR3 is replaced by an Arg in humNE. 16,17 We have designed and synthesized selective peptidyl-diphenyl phosphonate inhibitors based on these structural differences between humPR3 and humNE using the sequence of an optimized peptide substrate of PR3. 18 Phosphonate inhibitors are peptide based transition state irreversible inhibitors which form transition-state-resembling complexes with serine proteases. ...
The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4-S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnVP(O-C6H4-4-Cl)2 enhanced the second order inhibition constant kobs/[I] towards PR3 by more than ten times (kobs/[I] = 73.000 ± 5.000 M-1s-1) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.
... In CoMFA analysis, each molecule is located within fixed lattice points, and the intrinsic assumption is that each molecule interacts with the target receptor in all directions, which means that the whole molecule interacts with the receptor. However, according to X-ray crystallography of the protein-ligand complex, some parts of each molecule do not interact with receptor (Bode et al. 1986). These parts are considered as the noise of CoMFA analysis. ...
A comparative molecular field analysis has been developed to study the three-dimensional quantitative structure–activity relationship of a series of triterpene-based γ-secretase modulators. We have performed the genetic algorithm on a large set of comparative molecular field analysis fields to select the most responsible fields contributing to inhibitory activities of these compounds against Alzheimer’s disease. The genetic algorithm-selected comparative molecular field analysis fields were introduced into the partial least squares and principal component analysis to reduce the dimensionality of the input features. The extracted partial least squares components were used as inputs to build partial least squares regression (genetic algorithm-partial least squares regression), and the extracted principal components were used as inputs for principal component regression (genetic algorithm-principal component regression) and support vector regression (genetic algorithm-principal component analysis-support vector regression). The classic three-dimensional quantitative structure–activity relationship comparative molecular field analysis analysis (partial least squares regression) is also carried out for the sake of comparison. The results show that among the constructed models, in terms of root mean squares and leave-one-out cross-validated R²(q²), the combination of principal component analysis and support vector machine can effectively improve the prediction performance (RMSEtrain = 0.231, RMSEtest = 0.360, and q² = 0.638) compared with PLSR (RMSEtrain = 0.415, RMSEtest = 0.680, and q² = 0.311). The performances of the genetic algorithm-principal component regression and genetic algorithm-partial least squares regression were also comparable but less powerful than genetic algorithm-principal component analysis-support vector regression. Finally, based on the information derived from the comparative molecular field analysis contour map, some key features for increasing the activity of γ-secretase modulators have been identified to design new triterpene-based Alzheimer’s disease drugs.
... The crystal structure of uncomplexed recombinant PR3 (1FUJ) produced in insect cells was identified by molecular replacement using the structure of crystallized HNE (Fujinaga et al., 1996). Several threedimensional structures of HNE complexed with the third domain of turkey ovomucoid (Bode et al., 1986), domain 2 of SLPI (Koizumi et al., 2008), and synthetic inhibitors have all been determined (Wei et al., 1988;Navia et al., 1989;Huang et al., 2008;Hansen et al., 2011;Lechtenberg et al., 2015). PR3 and HNE share a high sequence identity (56%) and display a common fold. ...
Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener’s granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.
... The catalytic residues are located at the junction of the β-barrels. The active site of the NE runs along the β-barrels perpendicular to the catalytic amino acids (507). A list of biologically relevant NE substrates is presented in Table 3. ...
Elafin, an endogenous serine protease inhibitor, is a critical component of the epithelial barrier against neutrophil elastase (NE) activity. The central hypothesis examined in this dissertation was that elafin has tumor suppressive properties in breast cancer. In support of this hypothesis, immunohistochemical (IHC) analysis revealed that elafin was downregulated in the majority of invasive breast tumors and a subset of pre-invasive ductal carcinoma in situ (DCIS) compared to elafin expression in the normal mammary epithelium. To understand the role of elafin in the mammary epithelium and the impetus for its downregulation during breast tumorigenesis, primary and immortalized human mammary epithelial cells (HMECs) were utilized as a model system. Elafin was highly expressed in G0-arrested HMECs, suggesting a previously unrecognized role for elafin in growth control. Stable knockdown (KD) of elafin compromised the ability of HMECs to maintain G0-arrest during long-term growth factor deprivation. This effect was reversed by re-expression of wild-type elafin but not elafin-M25G lacking protease inhibitory function, suggesting a role for deregulated protease activity. Elafin KD HMECs demonstrated enhanced sensitivity to NE-induced proliferation. Mechanistically, activation of the ERK signaling pathway downstream of toll-like receptor 4 (TLR4) was essential to the mitogenic effect of NE in this system. Compared to HMECs, the majority of breast cancer cell lines lack endogenous elafin expression. Adenoviral-mediated expression of elafin was utilized to evaluate the tumor suppressive properties of elafin in breast cancer cell lines. Rb-status was identified as the critical factor governing the anti-tumor effect of elafin in this system. In breast cancer cell lines expressing functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. However, in breast cancer cell lines lacking functional Rb, elafin expression induced caspase-3 dependent apoptotic cell death. Elafin is a critical counterbalance against NE-activity. IHC analysis revealed that high levels of NE-expressing tumor-associated neutrophils (TAN) were associated with reduced recurrence-free survival, high tumor grade, and triple-receptor negative breast cancer (TNBC). ERK-catalyzed phosphorylation of p90RSK (T359/S363) and Rb phosphorylation (S807/811) were significantly enriched in NE-positive breast tumors, suggesting that NE-induced ERK signaling and proliferation may be relevant to human breast cancer. The in vivo role of deregulated NE in breast tumorigenesis was examined in the C3(1)TAg mouse model of TNBC. Knockout of NE in C3(1)TAg mice significantly reduced tumor growth and proliferation. Elafin has tumor suppressive properties in the context of breast cancer and is a critical counterbalance against the growth promoting effect of NE in vitro and in vivo. Deregulated NE-activity is a viable therapeutic target in breast cancer.
... In designing scaffolds I, the X-ray crystal structure of the HNE-TOMI complex 25 (PDB 1HNL) was utilized. In this complex, the protein inhibitor occupies an extended binding region stretching from S 5 to S 3 ', corresponding to residues -Pro-Ala-Cys-Thr-Leu-Glu-Tyr-Arg-of the inhibitor (Figure 6). ...
Neutrophil elastase (NE) is a proteolytic enzyme released extracellular during the formation of neutrophil extracellular traps (NETs) through degranulation. In addition to participating in the body's inflammatory response, NE also plays an important role in cancer. It can promote tumor proliferation, migration, and invasion, induce epithelial-mesenchymal transition (EMT), and change the tumor microenvironment (TME) to promote tumor progression. Concurrently, NE promotes systemic treatment resistance by inducing EMT. However, it can also selectively kill cancer cells and attenuate tumor development. Sivelestat is a specific NE inhibitor that can be used in the perioperative period of esophageal cancer patients to reduce the incidence of postoperative complications after esophagectomy. In addition, the combination of sivelestat and trastuzumab can enhance the efficacy of human epidermal growth factor receptor 2(HER 2) positive breast cancer patients. Meanwhile, targeting the human antibody domains and fragments of NE is also a new way to treat cancer and inflammation-related diseases. This review provides valuable insights into the role of NE in cancer treatment. Additionally, we discuss the challenges associated with the clinical application of sivelestat. By shedding light on the promising potential of NE, this review contributes to the advancement of cancer treatment strategies.
Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.
A growing number of computational tools have been developed to accurately and rapidly predict the impact of amino acid mutations on protein-protein relative binding affinities. Such tools have many applications, for example, designing new drugs and studying evolutionary mechanisms. In the search for accuracy, many of these methods employ expensive yet rigorous molecular dynamics simulations. By contrast, non-rigorous methods use less exhaustive statistical mechanics, allowing for more efficient calculations. However, it is unclear if such methods retain enough accuracy to replace rigorous methods in binding affinity calculations. This trade-off between accuracy and computational expense makes it difficult to determine the best method for a particular system or study. Here, eight non-rigorous computational methods were assessed using eight antibody-antigen and eight non-antibody-anti-gen complexes for their ability to accurately predict relative binding affinities (ΔΔG) for 654 single mutations. In addition to assessing accuracy, we analyzed the CPU cost and performance for each method using a variety of physico-chemical structural features. This allowed us to posit scenarios in which each method may be best utilized. Most methods performed worse when applied to antibody-antigen complexes compared to non-antibody-antigen complexes. Rosetta-based JayZ and EasyE methods classified mutations as destabilizing (ΔΔG <-0.5 kcal/mol) with high (83-98%) accuracy and a relatively low computational cost for non-antibody-antigen complexes. Some of the most accurate results for antibody-anti-gen systems came from combining molecular dynamics with FoldX with a correlation coefficient (r) of 0.46, but this was also the most computationally expensive method. Overall, our results suggest these methods can be used to quickly and accurately predict stabilizing versus destabilizing mutations but are less accurate at predicting actual binding affinities. This study highlights the need for continued development of reliable, accessible, and reproducible methods for predicting binding affinities in antibody-antigen proteins and provides a recipe for using current methods.
A growing number of computational tools have been developed to accurately and rapidly predict the impact of amino acid mutations on protein-protein relative binding affinities. Such tools have many applications, for example, designing new drugs and studying evolutionary mechanisms. In the search for accuracy, many of these methods employ expensive yet rigorous molecular dynamics simulations. By contrast, non-rigorous methods use less exhaustive statistical mechanics, allowing for more efficient calculations. However, it is unclear if such methods retain enough accuracy to replace rigorous methods in binding affinity calculations. This trade-off between accuracy and computational expense makes it difficult to determine the best method for a particular system or study. Here, eight non-rigorous computational methods were assessed using eight antibody-antigen and eight non-antibody-antigen complexes for their ability to accurately predict relative binding affinities (ΔΔG) for 654 single mutations. In addition to assessing accuracy, we analyzed the CPU cost and performance for each method using a variety of physico-chemical structural features. This allowed us to posit scenarios in which each method may be best utilized. Most methods performed worse when applied to antibody-antigen complexes compared to non-antibody-antigen complexes. Rosetta-based JayZ and EasyE methods classified mutations as destabilizing (ΔΔG < -0.5 kcal/mol) with high (83-98%) accuracy and a relatively low computational cost for non-antibody-antigen complexes. Some of the most accurate results for antibody-antigen systems came from combining molecular dynamics with FoldX with a correlation coefficient (r) of 0.46, but this was also the most computationally expensive method. Overall, our results suggest these methods can be used to quickly and accurately predict stabilizing versus destabilizing mutations but are less accurate at predicting actual binding affinities. This study highlights the need for continued development of reliable, accessible, and reproducible methods for predicting binding affinities in antibody-antigen proteins and provides a recipe for using current methods.
Human neutrophil elastase (hNE) is a serine protease that plays a major role in defending the bacterial infection. However, elevated expression of hNE is reported in lung and breast cancer, among others. Moreover, hNE is a target for the treatment of cardio pulmonary diseases. Ecotin (ET), is a serine protease inhibitor present in many gram-negative bacteria and it has a physiological role in inhibiting host proteases including hNE. Despite this known interaction, the structure of hNE-ET complex has not been reported, and the mechanism of ecotin inhibition is not available. We solved the structure of hNE-ET complex by molecular replacement method. The structure of the hNE-ET complex revealed the presence of six interface regions comprising of 50’s, 60’s and 80’s loops, between ET dimer and two independent hNE monomers, which explains the high affinity of ecotin to hNE (12pM). Notably, we observed a secondary binding site of hNE located 24 Å away from the primary binding site. Comparison of the closely related trypsin-ecotin complex with our hNE-ET complex shows movement of the backbone atoms of the 80s and 50s loops by 4.6 Å, suggesting the flexibility of these loops to inhibit a range of proteases. Through a detailed structural analysis, we demonstrate the flexibility of the hNE subsites to dock various sidechains concomitant with inhibition, indicating the broad specificity of hNE against various inhibitors. These findings will aid in the design of chimeric inhibitors that target both sites of hNE and in the development of therapeutics to control hNE-mediated pathogenesis.
Whilst the incorporation of water occurs in many biomolecular interfaces, the role they play is poorly understood with little attention paid to their contribution in dictating the specificity of an interaction. To investigate this the Src Homology 2 (SH2) domain of the viral Src protein kinase and its interactions with various phosphotyrosyl peptides and peptidomimetic ligands was studied. X-ray analysis shows that a feature of SH2 domains is the involvement of water molecules in the peptide binding-site. SH2 domains play a fundamental role in signal transduction and are therapeutic drug targets. Changes in water molecule content (incorporation, removal) and/or effects on binding affinity of the biomolecular complexes are examined using a combination of the thermodynamic isothermal titration calorimetry (ITC) and nanoflow electrospray ionisation mass spectrometry (ESI-MS) techniques and correlated to known structural information. The results from this study are used to predict the nature of possible water-mediated binding in the SH2 domain of Fyn using similar ligands. The role of water in binding interactions is further investigated by applying an empirical relationship based on the correlation of solvent-accessible surface area burial and changes in heat capacity (ΔCp). The experimental ΔCp is determined using ITC for the SH2/ligand interactions. The effects of proton linkage on binding are considered and five different surface area-based models are tested (relating to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations). This allows the calculation of a range of thermodynamic state functions (ΔCp, ΔS, ΔH and ΔG) directly from structure. Comparison with the experimentally derived data shows little agreement for the observed trends in the interactions of selected phosphotyrosyl peptides and SrcSH2. Furthermore, the different models have a dramatic effect on the calculated thermodynamic functions, thus binding energies predicted from these types of correlations are highly model dependent.
Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2‐macroglobulin (α2‐M), serpins [α1‐proteinase inhibitor (α1‐PI)], monocyte neutrophil elastase inhibitor (MNEI), α1‐antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1‐PI and MNEI but not by SLPI. α2‐M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2‐M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2‐M and hPR3 is governed by a kass in the ≤ 10⁵ m⁻¹·s⁻¹ range. Since α2‐M‐trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2‐M bait region (residues 690–728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39‐residue bait region of α2‐M (39pep‐α2‐M). Since the 39pep‐α2‐M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2‐M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well‐recognized function of major target autoantigen in granulomatosis with polyangiitis.
Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizing the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-ValP(OPh)2] for potency and selectivity. A series of adaptations both at the warhead and specificity residues P1 and P3 yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin inhibition assays were also conducted to measure off-target selectivity. Replacing the phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P3 (8a) yielded approximately 33-fold increase in CtHtrA inhibitory activity, with an IC50 = 0.68 ± 0.02 µM against HNE, while valine at P1 retained the best anti-chlamydial activity. This study provides a pathway for obtaining clinically relevant inhibitors.
As with proteins and nucleic acids, the structure and dynamics of oligosaccharides can be quite important to their biological function. Many oligosaccharides, such as the carbohydrate portions of glycoproteins, are involved in molecular recognition, in which their particular structures and conformations are recognized by other proteins which specifically bind to them. Furthermore, since many of the disaccharides are the repeat units of polysaccharides, their dynamical behavior and conformational preferences largely shape the properties of these larger polymers as well. For these reasons, it would be most desirable to develop a general understanding of oligosaccharide conformations and dynamics. While a great deal is known about the primary structures of oligosaccharides (Doubet et al., 1989), it has unfortunately proven to be quite difficult to study their conformations and dynamics experimentally, and this class of molecules is generally much more poorly known than other types of biopolymers.
Les protéases sont impliquées dans un grand nombre de processus physiologiques et pathologiques. En particulier, dans la mucoviscidose, un déséquilibre local entre les protéases leucocytaires et leurs inhibiteurs naturels, l'alpha-1-inhibiteur de protéase et l'inhibiteur bronchique, va être impliqué dans la destruction lente de la trame élastique du poumon. La mucoviscidose se caractérise, au niveau pulmonaire, par une accumulation de mucus purulent, une obstuction des voies aériennes et une inflammation conduisant à une détresse respiratoire et finalement à la mort. Au cours de l'inflammation, le neutrophile va libérer différentes protéases, suite à une phagocytose excessive ou après la mort cellulaire. D'un autre côté, la quantité importante d'ADN dérivée de ces neutrophiles augmante la viscosité des sécrétions bronchiques. Le présent travail vise à montrer le rôle de l'ADN dans l'inhibition de l'élastase leucocytaire et des autres protéases contenues dans les granules azurophiles du neutrophile, la cathepsine G et la protéinase 3.
Nous avons montré que l'ADN était capable de se fixer à la cathepsine G, à l'élastase leucocytaire et à l'inhibiteur bronchique. L'ADN est un inhibiteur hyperbolique de ces deux protéases, entraînant une inactivation de l'ordre de 90% de leur activité élastolytique. L'ADN modifie également fortement les interactions entre l'élastase et l'inhibiteur bronchique, en favorisant la dissociation du complexe. D'autres polynucléotides ont été étudiés. Parmi ceux-ci, l'ARNt et le polydC(24-36) sont également des inhibiteurs hyperboliques de l'élastase et de la cathepsine G. Ils favorisent tous les deux la dissociation du complexe élastase-inhibiteur bronchique. L'ADN modifie peu l'interaction entre l'élastase et l'alpha-1-inhibiteur de protéase, par contre l'ARNt et le polydC(24-36) entraînent une baisse de l'association, avec l'apparition d'un intermédiaire réactionnel. Il a également été montré que la rhDNAse ne levait pas l'inhibition de l'élastase et de la cathepsine G par l'ADN lorsque les complexes étaient déjà formés. Des mesures effectuées sur des sécrétions de patients atteints de mucoviscidose semblemt confirmer ce résultat.
En plus de ses manifestations pulmonaires, la mucoviscidose conduit à des dérèglements du pancréas exocrine. Il a été montré que l'inhibiteur bronchique est un bon inhibiteur de la chymotrypsine A, de même que l'égline C qui inhibe également l'élastase pancréatique, tandis que l'aprotinine est un bon inhibiteur des trypsines 1 et 2. L'inhibiteur bronchique étant clivé par la pepsine, la fraction qui atteint l'estomac après traitement de patients atteints de mucoviscidose n'atteint pas le duodénum dans sa forme active et n'aggrave donc pas leur insuffisance pancréatique.
The development of docking algorithms to predict near-native structures of protein:protein complexes from the structure of the isolated monomers, is of paramount importance for molecular biology and drug discovery. In this study, we assessed the capacity of the interfacial area of protein:protein complexes and of Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA)-derived properties, to rank docking poses. We used a set of 48 protein:protein complexes, and a total of 67 docking experiments distributed among bound:bound, bound:unbound, and unbound:unbound test cases. The MM-PBSA binding free energy of protein monomers, has been shown to be very convenient to predict high-quality structures with a high success rate. In fact, considering solely the top-ranked pose of more than 200 docking solutions of each of 39 protein:protein complexes, the success rate was of 77% in the prediction of high-quality poses, or 90% if considering high- or medium-quality poses. If considering high- or medium-quality poses in the top-one prediction, a success rate of 87% was obtained for a scoring scheme based on computational alanine scanning mutagenesis data. Such ranking accuracy highlights the ability of these properties to predict near-native poses in protein:protein docking.
Determination of protein-protein binding affinity values is key to understanding various underlying biological phenomena, such as how mutations change protein-protein binding. Most existing non-rigorous (fast) and rigorous (slow) methods that rely on all-atom representation of the proteins force the user to choose between speed and accuracy. In an attempt to achieve balance between speed and accuracy, we have combined rigorous umbrella sampling molecular dynamics simulation with a coarse-grained protein model. We predicted the effect of mutations on binding affinity by selecting three protein-protein systems and comparing results to empirical relative binding affinity values, and to non-rigorous modeling approaches. We obtained significant improvement both in our ability to discern stabilizing from destabilizing mutations and in the correlation between predicted and experimental values compared to non-rigorous approaches. Overall our results suggest that using a rigorous affinity calculation method with coarse-grained protein models could offer fast and reliable predictions of protein-protein binding free energies.
JO146, a mixture of two diastereomers of a peptidic phosphonate inhibitor for Chlamydial HtrA (CtHtrA), has reported activity against Chlamydia species in both human and koala. In this study we isolated the individual diastereomers JO146-D1 and JO146-D2 (in ≥90% purity) and assessed their individual inhibitory activity against the serine protease human neutrophil elastase (HNE) which is structurally and functionally related to CtHtrA, as well as in Chlamydia trachomatis cell culture. JO146-D2 [S,S,R-Boc-Val-Pro-ValP(OPh)2], the isomer with the physiologically relevant valine at P1, had an approximate 2.5 - fold increase in in vitro HNE inhibition potency over JO146-D1 [S,S,S-Boc-Val-Pro-ValP(OPh)2] and greater than 100 - fold increase in cellular anti-chlamydial activity compared to JO146-D1 which possesses the unnatural valine at P1. JO146 and the individual diastereomers had excellent selectivity for the serine protease HNE over the potential off-target serine proteases trypsin and chymotrypsin. Docking studies supported the biological data with a geometrically unfavoured interaction observed between the P1 valine residue of JO146-D1 and the enzyme S1 sub-pocket.
http://hdl.handle.net/10523/12122
Our Fuzzy-Border (FB) continuum solvent model has been extended and modified to produce hydration parameters for small molecules using POlarizable Simulations Second-order Interaction Model (POSSIM) framework with an average error of 0.136 kcal/mol. It was then used to compute pKa shifts for carboxylic and basic residues of the turkey ovomucoid third domain (OMTKY3) protein. The average unsigned errors in the acid and base pKa values were 0.37 and 0.4 pH units, respectively, versus 0.58 and 0.7 pH units as calculated with a previous version of polarizable protein force field and Poisson Boltzmann continuum solvent. This POSSIM/FB result is produced with explicit refitting of the hydration parameters to the pKa values of the carboxylic and basic residues of the OMTKY3 protein; thus, the values of the acidity constants can be viewed as additional fitting target data. In addition to calculating pKa shifts for the OMTKY3 residues, we have studied aspartic acid residues of Rnase Sa. This was done without any further refitting of the parameters and agreement with the experimental pKa values is within an average unsigned error of 0.65 pH units. This result included the Asp79 residue that is buried and thus has a high experimental pKa value of 7.37 units. Thus, the presented model is capable or reproducing pKa results for residues in an environment that is significantly different from the solvated protein surface used in the fitting. Therefore, the POSSIM force field and the FB continuum solvent parameters have been demonstrated to be sufficiently robust and transferable. © 2016 Wiley Periodicals, Inc.
Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS) in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML) techniques. Our model is trained on a large number of complexes and on a significantly larger number of different structural- and evolutionary sequence-based features. In particular, we added interface size, type of interaction between residues at the interface of the complex, number of different types of residues at the interface and the Position-Specific Scoring Matrix (PSSM), for a total of 79 features. We used twenty-seven algorithms from a simple linear-based function to support-vector machine models with different cost functions. The best model was achieved by the use of the conditional inference random forest (c-forest) algorithm with a dataset pre-processed by the normalization of features and with up-sampling of the minor class. The method has an overall accuracy of 0.80, an F1-score of 0.73, a sensitivity of 0.76 and a specificity of 0.82 for the independent test set.
Serine and cysteine proteases play a central role in digestion, protein degradation and in bioregulation. Their activity may be controlled by activation from inactive proenzyme forms and by inhibition by natural inhibitors. Detailed structural studies have provided a basis for understanding the principles of activation, activity and inhibition of proteases. In serine proteases the functional significance of molecular flexibility was first documented (Huber & Bode, 1978; Walter et al, 1982). The basic pancreatic trypsin inhibitor BPTI served as model to develop biophysical and biochemical techniques (like protein NMR spectroscopy (Wiithrich, 1989), hydrogen-deuterium exchange (Wagner & Wiithrich, 1982), experimental folding studies (Creighton & Goldenberg, 1984) and theoretical methods (like molecular dynamics (Karplus & McCammon, 1981; Van Gunsteren et al, 1983) and normal mode analysis (Levitt et al., 1985)). Its polypeptide chain fold is shown in Figure 1.
Molecular recognition processes depend on the complementarity in shape and physical chemical properties of the surface of the molecules or macromolecules which interact. While generally accepted, this general statement has little heuristic value, for it does not quantify the role of hydrophobicity, H-bond forming capacity, electric charge distribution and other relevant properties. We present here an analysis of specific protein-protein complexes for which detailed atomic structures are available, and we show that shape complementarity is a valid criterion for a first selection of interacting surfaces on macromolecules.
Human leukocyte elastase (HLE) is a major protein of the azurophilic granules of human polymorphonuclear granulocytes (Fig.1). It is believed to be normally involved in the intracellular degradation of cellular and phagocytosed proteins and bacteria. Upon stimulation it is, however, released into extracellular space: into the plasma, where it is normally blocked by the α1-proteinase inhibitor (α1PI), and into mucous secretions, where it can be inhibited by the mucous proteinase inhibitor (MPI).
Chronic Obstructive Pulmonary Disease (COPD), comprised of chronic bronchitis and pulmonary emphysema, is a serious health problem in the world. In the USA, COPD caused more than 70,000 deaths in 1986 and more than 10,000,000 Americans suffered from the disease.1 The association between severe α1protease inhibitor (α1PI) deficiency and emphysema has led to the hypothesis that an elastase-antielastase imbalance causes emphysema.2,3 Human neutrophil elastase (HNE) is most likely the cause of emphysema in both smokers and non-smokers, although the evidence supporting the elastase-antielastase hypothesis is largely indirect in smokers with normal protective levels of α1PI. Homogenates of leucocytes as well as highly purified preparations of HNE produce emphysema in experimental animals and only elastolytic enzymes will induce experimental emphysema. In addition, neutrophils are increased 4–5 fold in the lungs of smokers and it is postulated that α1PI is inactivated by powerful oxidizing agents in the cigarette smoke and PMNs contributing to the elastase-antielastase imbalance. Other proteases including human proteinase 3 and macrophage elastase may also play a role in lung destruction in emphysema. Nevertheless, the majority of investigators in the field believe that a human neutrophil elastase-antielastase imbalance plays the major role in the pathogenesis of emphysema.
α-Thrombin is a glycosylated trypsinlike serine proteinase (Magnusson, 1971), generated in the penultimate step of the blood coagulation cascade from the circulating plasma protein prothrombin. Upon autocatalytic and factor Xa cleavage, the functional two-chain molecule α-thrombin is generated. In the case of the human species this consists of the 36-residue A chain and the 259-residue B chain (Butkowski et al., 1977; Thompson et al., 1977; Walz et al., 1977; Degen et al., 1983). The two chains are covalently connected by a disulfide bridge. The B chain carries an asparagine-linked sugar chain (Fenton et al., 1977a; Butkowski et al., 1977); it has also been shown to be homologous to the catalytic domains of other pancreatic and coagulation/fibrinolytic trypsinlike proteinases (Jackson and Nemerson, 1980). Upon further autolytic or proteolytic cleavage, more species (in particular β- and γ-thrombin) are generated which retain some activity against small synthetic substrates, but have lost most or all clotting activity (Lundblad et al., 1979; Fenton, 1981, 1986; Berliner, 1984; Elion et al., 1986; Hofsteenge et al., 1988).
It is commonly acknowledged that neutrophil elastase (NE) is responsible for lung elastin degradation that leads to emphysema.
Human leukocyte elastase (HLE) is a serine protease and a basic glycoprotein, with isoforms of 25-30 kD. The active site of HLE is a channel on the protein surface that has extended substrate binding sites. The catalytic triad of Serlg5, Hisr7, and Asplo2 that resides in the active site channel catalyzes amide bond hydrolysis of various proteins, including elastin, the connective tissue of the lung. The S1 specificity pocket of serine proteases is largely responsible for the selectivity of the class of enzymes to cleave proteins at specific sites. HLE has a relatively small S1 pocket that is lined, with hydrophobic residues, thus HLE preferentially cleaves proteins at sites, with small lipophilic residues, such as alanine and valine. The specificity has been exploited in the design of HLE inhibitors. The isoforms of HLE have identical amino acid sequences and catalytic properties, but differ based on the nature of the carbohydrate content. The different carbohydrate content of the E-1 and E-3 isoforms of elastase is proposed to direct these isoforms to secretory and lysosomal functions, respectively. Human elastase, from polymorphonuclear neutrophils (PMN) and from purulent sputum, display identical kinetics, with various substrates and inhibitors, suggesting that elastase, from sputum, is from polymorphonuclear leukocytes (PMNs). In the chapter, the term HLE will be used to indicate elastase, from either PMNs or purulent sputum.
We have created models to predict cleavage sites for several human proteases including: caspase-1, caspase-3, caspase-6, caspase-7, cathepsin B, cathepsin D, cathepsin G, cathepsin K, cathepsin L, elastase-2, granzyme A, granzyme B, matrix metallopeptidase-2 (MMP2), MMP7, MMP9, thrombin and trypsin-1. Rather than representing the sequence pattern around the potential cleavage site through a series of flags with each flag representing one of the 20 standard amino acids, we first represent each amino acid by its calculated properties. For these calculated properties, we use validated cheminformatic descriptors, such as molecular weight, logP, and polar surface area, of the individual amino acids. Finally, the cleavage site specific descriptors are calculated through various combinations of the individual amino acid descriptors for the residues surrounding the cleavage site. Some of these combinations do not take into account the location of the residue, as long as it is in a prescribed neighborhood of the potential cleavage site, whereas others are sensitive to the precise order of the residues in the sequence. The key advantage of this approach is that it allows one to perform meaningful calculations with nonstandard amino acids for which little or no data exists. Finally, using both docking and molecular dynamics simulations, we examine the potential for and limitations of protease crystal structures to impact the design of proteolytically stable peptides. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
Proteinases play an important role in the normal functioning of biological systems. They are involved in crucial processes such as food digestion, blood coagulation and fibrinolysis, blood pressure regulation, and fertilization. Certain proteinases release peptide hormones and neuromodulators from inactive precursors or degrade message-transmitting peptides, thus initiating or terminating a variety of biological responses. It is clear that besides a pronounced cleavage specificity, a tight regulation of proteolytic activity by endogenous proteinase inhibitors is necessary. The number of proteinaceous inhibitors that have been isolated and characterized so far is large and growing steadily. The majority are directed toward members of the serine proteinases, one of the four classes of endopeptidases. Serine proteinase inhibitors can be grouped into at least sixteen different inhibitor families on the basis of sequence similarity, topological relationships between the disulfide bridges, and the location of the binding site for the cognate proteinase. Detailed knowledge of the structure and reactivity of the inhibitors is indispensable for a thorough understanding of the controlling functions they exercise in a variety of fundamental physiological proteolytic processes. This chapter discusses these inhibitors and some approaches that might lead to satisfactory therapeutic compounds.
Of the 85 three-dimensionally characterized residues of cytochrome b5, 51 are found to be structurally and topologically equivalent to the globin fold. When these proteins have been superimposed, the heme irons are found to be less than 1.4 A separated and the heme normals are inclined by less than 9.5 degrees. The proximal histidine of the globins and two adjacent helices are equivalent to the sixth iron ligand and adjacent helices of cytochrome b5. Larger differences in structure are observed on the distal side of the heme, coincident with the most changeable part of the globin structures. The heme itself is rotated by 53 degrees about its normal but such a change is energetically minimal and conservative as the heme side groups are not directly involved in the function of the molecules. The beta-sheet of cytochrome b5 is inserted into a corresponding cavity of the globins forming an additional lining to the heme pocket. The roughly 50 residues missing at the carboxy end of the known cytochrome b5 fragment could correspond in part to the H helix in the globins. While it would seem probable that these similarities represent divergent evolution from a primordial heme-binding protein, the possibility of structural convergence to a functionally satisfactory protein cannot be excluded.
The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R-value of 0.18 (8-1.2 A). Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.
We have determined the crystal structure of the molecular complex between Streptomyces griseus protease B (SGPB), a bacterial serine protease, and the third domain of the ovomucoid inhibitor from turkey. Restrained-parameter least-squares refinement of the structure with the 1.8-A intensity data set has resulted in an R factor of 0.125. The carbonyl carbon atom of the reactive bond between Leu-18 and Glu-19 in the inhibitor lies at a distance of 2.71 A from the O gamma atom of the nucleophilic Ser-195 in SGPB; this distance is 0.5 A shorter than a normal van der Waals contact. Unlike the reactive bond in the pancreatic trypsin inhibitor complexed with bovine trypsin, the Leu--Glu bond of the ovomucoid inhibitor is not distorted from planarity towards a pyramidal configuration.
Japanese quail ovomucoid exists in two polymorphic forms. One has serine, the other glycine at position 162. The tryptic peptide corresponding to positions 160 to 164 was purified from ovomucoids isolated from egg whites of eggs laid by 11 different hens and subjected to amino acid analysis. The quantitative distribution of serine and glycine in this pentapeptide is consistent with the interpretation that the ovomucoid gene exists in two codominant allelic forms at one locus. Even though the gene product is apparently expressed only in the female, these results indicate that the ovomucoid structural gene is transmitted as a simple Mendelian character which is neither sex-linked nor shows dominance. Intact third domains (positions 131 to 186) isolated from the two allelic forms of ovomucoid interact with bovine beta-trypsin in a similar but not identical manner; the complex with the glycine form dissociates more rapidly. Evidence is presented which suggests that glycine is the ancestral residue at position 162; yet, the serine form is the more frequent phenotype.
The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americans), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. α-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.
Three isomorphous heavy-atom derivatives have been used to calculate a 2.5 Å resolution electron density map of tosyl-elastase at pH 5.0, from which an accurate atomic model has been constructed. Atomic co-ordinates measured from this model have been refined using model building, real-space refinement and energy minimization programs. The three-dimensional conformation of the polypeptide chain is described in terms of conformational angles, hydrogen-bonding networks and the environment of different types of amino acid side-chain.Difference Fourier calculation of the high resolution structure of native elastase at pH 5.0 shows it to be virtually identical to that of the tosyl derivative, except near the tosyl group. The conformation of the catalytically important residues in native elastase is very similar to that of native α-chymotrypsin, except for the orientation of the active centre serine oxygen. The significance of important structural similarities and differences between these two enzymes is discussed.Elastase contains 25 internal water molecules which play an important role in stabilizing the active conformation of the enzyme. Many of these water molecules are in identical positions to those found in the interior of α-chymotrypsin
The crystal structure of benzamidine-inhibited bovine β-trypsin has been refined by constrained crystallographic refinement at 1·8 Å resolution. The final R-value is 0·23. The estimated standard deviation of the atomic co-ordinates is less than 0·1 Å. A preliminary refinement of inhibitor-free trypsin at 1·5 Å resolution shows only very slight conformational changes to benzamidine-inhibited β-trypsin.The differences between internal main-chain atoms of free trypsin and the trypsin component in the trypsin-inhibitor complex (Huber et al., 1974a) is 0·26 Å, <Δø}, <Δψ> are 8·0°. Complex formation does not significantly influence the peptide chain folding and only slightly alters the active site residues. In particular, Ser 195 Oγ does not change its dihedral angle considerably.The refinement led to the finding of a single site occupied by calcium (Bode & Schwager, 1975). The peptide chain folding, hydrogen bond pattern and the location of internal solvent is very similar to free α-chymotrypsin (Birktoft & Blow, 1972). Ser 195 Oγ, however, differs by about 180° in dihedral angle.A comparison with the partial atomic co-ordinate list given by Stroud et al. (1974) and Krieger et al. (1974) for benzamidine-inhibited bovine trypsin shows large discrepancies in the specificity site Asp189, Ser190, Gln192 and the active site-associated solvent.
Diffraction data for α-chymotrypsin crystals at −10 °C were measured at 1·68 Å resolution and refined by restrained structure-factor least-squares refinement. The two independent chymotrypsin molecules in the crystallographic asymmetric unit were refined independently. The overall structure of α-chymotrypsin is little changed from published co-ordinates. The root-mean-square shift of Cα co-ordinates is 0·42 Å, co-ordinates for the two molecules showing a root-mean-square difference of 0·19 Å. Certain regions with high disorder (residues 9 to 14, 73 to 79) remain difficult to interpret and several side-chains are disordered. Some water molecule positions have been changed.The absence of the tosyl group has made a significant difference to the refined structure at the active site. This now agrees closely with other enzymes of the trypsin family that have been refined at high resolution. There is a strong hydrogen bond between Nε2 (His57) and Oγ (Ser195) in the free enzyme, in line with the published description of the charge relay system.
The complex formed by porcine pancreatic kallikrein A with the bovine pancreatic trypsin inhibitor (PTI) has been crystallized at pH 4 in tetragonal crystals of space group P41212 with one molecule per asymmetric unit. Its crystal structure has been solved applying Patterson search methods and using a model derived from the bovine trypsin-PTI complex (Huber et al., 1974) and the structure of porcine pancreatic kallikrein A (Bode et al., 1983). The kallikrein-PTI model has been crystallographically refined to an R-value of 0·23 including X-ray data to 2·5 Å.
"Group-specific" protease (GSP) is a serine protease, obtained from rat small intestine, which preferentially inactivates the apo forms of certain pyridoxal phosphate requiring enzymes. The enzyme contains 224 amino acid residues in a single polypeptide chain and three disulfide bonds. In the present work the covalent structure has been determined and its homologous relationship to those of chymotrypsin, trypsin, and elastase has been established (approximately 33% identity with each). The residues forming the "charge-relay" system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions in GSP, whereas an alanyl residue at position 176 of GSP corresponds to a residue which participates in the primary substrate binding site in serine proteases (Asp-177 in trypsin; Ser-189 in chymotrypsin). Three disulfide bonds in GSP occur in similar positions in chymotrypsin, trypsin, and elastase. However, GSP lacks a disulfide bond which is present in all known serine proteases (linking Cys-191 to Cys-220 in chymotrypsin). In view of the close proximity of this bond to both the primary and the antiparallel binding sites of various serine proteases, it is likely that its absence in GSP is related to the substrate specificity of this enzyme. It is concluded that GSP diverged from a common ancestor preceding chymotrypsin but following trypsin.
The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. alpha-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.
Human granulocytic elastases have been purified by a two-step procedure involving affinity chromatography of crude extracts of leukocytic granules on Sepharose-Trasylol, followed by ion-exchange chromatography on CM-cellulose to resolve the isoelastases. All of these enzymes were found to be glycoproteins with the carbohydrate content of the major form being composed essentially of only neutral sugars. The molecular weight of this form was found to be near 30 000 daltons with the other forms being slightly higher. Preliminary structural analyses indicate that all of the elastase isozymes have identical NH2-terminal sequences suggesting that the differences in mobility of the four proteins are not due to different degrees of activation from a common zymogen but, more likely, from minor changes in carbohydrate content. Human granulocytic elastases are less active on ligament elastin than porcine pancreatic elastase, but both are inhibited by synthetic elastase active-site directed low molecular weight compounds (Tuhy, P. M., and Powers, J. C. (1975), FEBS Lett. 50, 359) as well as by plasma alpha-1-proteinase inhibitor (formerly called alpha-1-antitrypsin). In the latter case a stable complex with mol wt of 78 000 daltons is formed indicating the formation of a 1:1 complex.
Human neutrophil elastase from normal donors has been purified using an isolation procedure which included sequential sodium chloride extraction, Aprotinin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil elastase and resulted in a higher specific activity of the final preparation. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of the reduced purified protein demonstrated three polypeptides of Mr 31,000, 28,000, and 27,500. Four polypeptides were resolved on acid gel electrophoresis; each of the four possessed amidolytic activity. Furthermore, peptide analysis of Staphylococcus aureus V8 protease digests indicated that these polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The apparent isoelectric points of these four forms as determined by two-dimensional electrophoresis range from 6.1 to 6.7. By utilizing microsequencing techniques, the first 40 residues of neutrophil elastase have been determined and compared with the reported sequence of elastase isolated from leukemic myeloid cells. In addition, a high degree of homology was found within the amino-terminal regions of neutrophil elastase and the serine proteinases porcine elastase, bovine chymotrypsin, human factor D, and the beta chain of plasmin.
Porcine leukocyte elastase was purified from granulocytes by chelating chromatography on copper chelate Sepharose and by ion exchange chromatography on CM-Sepharose. Thus an enzyme preparation with a specific activity (substrate: MeOSuc(Ala)2ProValNan) of 89.3 U/mg protein was obtained. Dodecyl sulphate gel electrophoresis revealed one protein band corresponding to a molecular mass of 27 kDa. The amino acid composition was determined and isoleucine was identified as the only N-terminal amino acid residue. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 2000 1 . mol-1 . min-1. The dissociation constants, Ki, of the complexes of porcine leukocyte elastase with various inhibitors were calculated. The kinetic constants for the elastase-catalysed hydrolysis of MeOSuc(Ala)2ProValNan, Suc(Ala)2ValNan and Suc(Ala)3Nan were determined, as well as the kinetic constants of the inactivation of leukocyte elastase by active site mapping reagents. Detergents such as Triton X-100, Tween 20 and Brij 35, as well as porcine serum albumin, activated the porcine leukocyte elastase preparation.
This article surveys elastinolytic proteinases in man, excluding enzymes of the pancreas and digestive tract. Special emphasis is placed on the elastase of polymorphonuclear neutrophils (PMN). The properties of this latter enzyme, its target molecules in plasma and connective tissues, and its endogenous regulators are briefly discussed. Persistent activity of the enzyme, even in the presence of its regulatory inhibitors, is explained. The chapter closes with a brief discussion of several pulmonary diseases in which elastase-mediated tissue injury is thought to play a role.
OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].
Elastolytic enzymes were isolated from the extract of the lysosome like granules of human leukemic myeloid cells by chromatography on Sephadex G 75, εAhx Sepharose 4 B, elastin Sepharose 4 B and preparative agarose gel electrophoresis. The elastase were cationic proteins but appeared as three proteins with a slight difference in electrophoretic mobility. The three elastases degraded native, insoluble elastin with a pH optimum of about 8.5. The granulocyte elastases were inactivated by α1 antitrypsin and α2 macroglobin. The amino acid composition of the three elastases was the same except for a variation in arginine content. Dodecylsulfate electrophoresis suggested molecular weights in the range 33000-36000. The s020(w) value determined for one of the elastases was 2.6 S. The three elastases gave the reaction of identity on immunodiffusion according to Ouchterlony and crossed immunoelectrophoresis. They showed the same type of reaction with elastase partly purified from normal human granulocytes, which also contained three separate elastolytic enzymes.
The crystal structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor has been refined with data to 1.9 Å resolution, using a procedure described by Deisenhofer & Steigemann (1974) in their refinement of the crystal structure of the free inhibitor. This procedure involves cycles consisting of phase calculation using the current atomic model, Fourier synthesis using these phases and the observed structure factor amplitudes and Diamond's real-space refinement (Diamond, 1971,1974). At various stages, difference Fourier syntheses are calculated to detect and correct gross errors in the model and to localize solvent molecules.The refinement progressed smoothly, starting with the model obtained from the isomorphous Fourier map at 2.6 Å resolution. The R-factor is 0.23 for 20,500 significantly measured reflections to 1.9 Å resolution, using an over-all temperature factor of 20 Å2. The estimated standard deviation of atomic positions is 0.09 Å.An objective assessment of the upper limit of the error in the atomic coordinates of the final model is possible by comparing the inhibitor component in the model of the complex with the refined structure of the free inhibitor (Deisenhofer & Steigemann, 1974). The mean deviation of main-chain atoms of the two molecular models in internal segments is 0.25 Å, of main-chain dihedral angles 5.1 ° and side-chain dihedral angles 6.5 °.A comparison of the trypsin component with α-chymotrypsin (Birktoft & Blow, 1972) showed a mean deviation of main-chain atoms of 0.75 Å. The structures are closely similar and the various deletions and insertions cause local structural differences only.
Two closely related crystal structures of alpha 1-proteinase inhibitor modified at the reactive site peptide bond Met358--Ser359 have been analysed. The crystal structure has been obtained from diffraction data at 3 A resolution, with phases originally from isomorphous replacement. The electron density map was substantially improved by cyclic averaging of the electron densities of the two crystal forms and allowed the chain to be traced in terms of the known chemical amino acid sequence. Energy restrained crystallographic refinement was initiated and resulted in conventional R-values of 0.251 for the tetragonal crystal form (6 to 3 A resolution) and 0.247 for the hexagonal crystal form (6 to 3.2 A resolution). The polypeptide chain is almost completely arranged in well-defined secondary structural elements: three beta-sheets and eight alpha-helices. The helices are preferentially formed by the first 150 residues. They are in proximity underneath sheet A. The chain ends Met358 and Ser359 of the nicked species are arranged in strands on opposite ends of the molecule indicating a major structural rearrangement upon modification of the intact inhibitor. It is suggested that the Met358 strand is in a different conformation removed from sheet A and approaches Ser359 in the intact inhibitor species. Glu342, which is exchanged by a lysine in the Z-variant is in a strategic position for such a rearrangement. The three carbohydrate chains of alpha 1-proteinase inhibitor have partly defined electron density close to their attachment sites at asparagine residues. The anti-thrombin and ovalbumin amino acid sequences can be accommodated in the alpha 1 inhibitor molecular structure. The intron-exon junctions of the ovalbumin and the alpha 1-proteinase inhibitor gene are all in surface loops of the mature protein.
Renin is an aspartyl proteinase that catalyses the first, and rate-limiting, step in the conversion of angiotensinogen to the hormone angiotensin II. The catalysis is highly specific, and plays an important physiological part in the regulation of blood pressure. For this reason inhibitors of renin are of potential value in the treatment of certain forms of hypertension. Although progress has been made in the design of inhibitors for clinical use by modification of angiotensinogen sequences, and as pepstatin analogues or with reduced peptide bonds, we have now provided the basis for a more rational approach by the use of interactive computer graphics techniques to build a three-dimensional model of renin. The model is based on the three-dimensional structure of endothia pepsin and the primary structure of mouse renin, which is very similar to that of the human enzyme. We show that renin may have a three-dimensional structure similar to that of other aspartyl proteinases.
1. Aspergillopeptidase B rapidly hydrolyses the -Leu18-Glu19-reactive site peptide bond in turkey ovomucoid third domain (OMTKY3) within the pH-range of 4.0-8.4. The reaction proceeds to equilibrium between OMTKY3 and its modified form with the reactive site peptide bond cleaved (OMTKY3). 2. The dependence of the equilibrium constant (Khyd) on pH indicates that hydrolysis of the reactive site peptide bond apparently does not perturb the pK-values of any preexistent ionizable groups in OMTKY3. 3. The obtained Khyd0 value indicates that free energies of OMTKY3 and OMTKY3 are essentially the same. 4. Hydrolysis of the reactive site peptide bond by aspergillopeptidase B at neutral pH is about 60 times faster than the same reaction catalyzed by subtilisin (Carlsberg), the enzyme strongly inhibited by OMTKY3. 5. Resynthesis of the reactive site peptide bond at neutral pH catalyzed by aspergillopeptidase B (reverse reaction) is almost four orders of magnitude faster than the forward reaction.
The structure of the complex between the serine protease Streptomyces griseus protease B (SGPB) and the third domain of the Kazal-type ovomucoid inhibitor from turkey has been solved at 1.8-A resolution and refined to a conventional R factor of 0.125. As others have reported previously for analogous complexes of proteases and protein inhibitors, the inhibitor binds in a fashion similar to that of a substrate; it is not cleaved, but there is a close approach (2.7 A) of the active site nucleophile Ser-195 O gamma to the carbonyl carbon of the reactive peptide bond of the inhibitor. Contrary to the structural reports regarding the other enzyme-inhibitor complexes, we conclude that there is no evidence for a significant distortion of this peptide bond from planarity. The mechanism of inhibition can be understood in terms of the equilibrium thermodynamic parameters Ka, the enzyme-inhibitor association constant, and Khyd, the equilibrium constant for inhibitor hydrolysis. These thermodynamic parameters can be rationalized in terms of the observed structure.
Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P41212 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure.
Japanese quail ovomucoid third domain (OMJPQ3), a Kazal-type inhibitor, was crystallographically refined with energy constraints. The final R-value is 0.20 at 1.9 Å resolution. The four molecules in the asymmetric unit are very similar, with deviations of main-chain atoms between 0.2 and 0.3 Å. An analysis of the side-chain hydrogen-bonding pattern and amino acid variability in the Kazal family shows a high correlation between hydrogen-bonding and conservation.The conformation of the reactive site loop (P2-P2′) of OMJPQ3 is similar to those of basic pancreatic trypsin inhibitor, Streptomyces subtilisin inhibitor, and soybean trypsin inhibitor. This suggests a common binding mode and justifies model-building studies of complexes.Complexes of OMJPQ3 with trypsin, chymotrypsin and elastase were modelled on the basis of the trypsin-basic pancreatic trypsin inhibitor complex structure and inspected by use of a computer graphics system. Stereochemically satisfying models were constructed in each case and detailed interactions are proposed. The complex with elastase is of particular interest, showing that leucine and methionine are good P1 residues. A good correlation is observed between functional properties of ovomucoid variants and the position of the exchanged residues with respect to the modelled inhibitor-protease contact.
Proteins have been classified into families based upon sequence homology. An accurate, systematic comparative model-building procedure for a homologous family of proteins would be very valuable scientifically. This paper presents such a procedure and applies it to the mammalian serine proteases, which are ubiquitous and involved in many important biological functions. Eleven proteins of this family are considered here, including a variety of blood serum, intestinal and pancreatic proteins as well as a closely related bacterial enzyme.The modeling method capitalizes upon the availability of three experimentally determined structures for mammalian serine proteases. These structures show that the molecule is divided into structurally conserved regions, which contain the strong sequence homology, and structurally variable regions, which include all the additions and deletions. We show that by applying this structural distinction to new sequences, erroneous alignments of the sequences are greatly minimized.For each aligned new sequence, the structurally conserved regions can be constructed from any of the known structures. In examining the variable regions, we have found that a variable region that has the same length and residue character in two different known structures usually has the same conformation in both. Thus, when the eight structurally unknown proteins are modeled, most of the variable regions can be constructed directly from the known structures. A minority of the variable regions require more sophisticated analysis to evaluate the relative merits of a small number of possible conformations. Only a very few are so different that modeling by homology is entirely ruled out. We demonstrate, therefore, that by this modeling procedure, the maximum of each of these mammalian serine proteases is constructed directly from the experimentally determined structures and the necessity to build from intuition or from energy considerations is greatly reduced.
The three-dimensional structure of the proteic complex formed by bovine trypsinogen and the porcine pancreatic secretory trypsin inhibitor (Kazal type) has been solved by means of Patterson search techniques, using a predicted model of the trypsin-ovomucoid complex (Papamokos et al., 1982). The structure of the complex, including 162 solvent molecules, has been refined at 1.8 Å resolution (26,341 unique reflections) to a conventional crystallographic R factor of 0.195. The inhibitor molecule binds to trypsinogen via hydrogen bonds and/or apolar interactions at sites P9, P7, P6, P5, P3, P1, P1′, P2′ and P3′ of the contact area. The structure of the inhibitor itself resembles closely that of the third domain of Japanese quail ovomucoid inhibitor, recently reported by Weber et al. (1981). The trypsinogen part of the complex resembles trypsin, as is the case in the trypsinogen-basic pancreatic trypsin inhibitor complex, but two segments of the activation domain adopt a different conformation. Most notably in the N-terminal region the Ile16-Gly19 loop, which is disordered in free trypsinogen and in the trypsinogen-basic pancreatic trypsin inhibitor complex (Huber & Bode, 1978), assumes a regular structure and the polypeptide chain can be traced as far as residue Asp14. This new and fixed structure allows the formation of a buried salt link between the side-chains of Lys156 and Asp194. Conformations differing from those of trypsin are also found for residues 20 to 28 and residues 141 to 155. Some structural perturbation is observed in other parts of the molecule, including the calcium loop.
The third domain of Japanese quail ovomucoid, a Kazal type inhibitor, has been crystallized and its crystal structure determined at 2.5 Å resolution using multiple isomorphous replacement techniques. The asymmetric unit contains four molecules. In the crystal the molecules are arranged in two slightly different octamers with approximate D4 symmetry. The molecules are held together mainly by interactions of the N-terminal residues, which form a novel secondary structural element, a β-channel.The molecule is globular with approximate dimensions 35 Å × 27 Å × 19 Å. The secondary structural elements are a double-stranded anti-parallel β-sheet of residues Pro22 to Gly32 and an α-helix from Asn33 to Ser44. The reactive site Lys18-Asp19 is located in an exposed loop. It is close to Asn33 at the N terminus of the helical segment. The polypeptide chain folding of ovomucoid bears some resemblance to other inhibitors in the existence of an anti-parallel double strand following the reactive site loop.
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