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Experimentally based model of the 16-kDa proteolipid. The helical organization within a 16-kDa proteolipid dimer was based on the differential distribution of functional/non-functional cysteine mutations (A) and proximity of cross-linking and suppressor pairs (B), as detailed in the " Discussion. " In A, the general disposition of functional mutations is indicated by the shaded areas. In B, crosslinking and suppressor pairs are connected by shaded and unshaded lines, respectively . Numbers indicate the residue positions, with the helices viewed perpendicular to the cytoplasmic surface of the membrane. Helices 1 and 3 have a high content of residues with low mass side chains, and are shown diagrammatically to have a smaller effective diameter than helices 2 and 4, according to Ref. 41. Helices 3 and 4 are shown displaced from the center of the dimeric complex solely for clarity.
Source publication
Theoretical mechanisms of proton translocation by the vacuolar H+-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior
surface of the membrane sector of the enzyme, contacting the lipid phase. However, structural support for this theoretical
mechanism is lacking. To address this,...
Contexts in source publication
Context 1
... Disulfide cross-linking data from this and previous stud- ies (20) indicate that helix 1 will line a pore at the center of this complex. The helices will be oriented with respect to each other such that Ser 25 of one monomer and Leu 27 of the neighboring polypeptide are sufficiently close that the they could cross-link when changed to cysteine (Fig. 9). Using the experimentally determined position of helix 1 as a foundation, the organization of the remaining transmembrane helices can be approximated on the basis of the functional impact of cysteine mutations. This approximation can then be refined by considering the relative positioning of cross-linking and suppressor pairs to ...
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... quence. However, sites within the proteolipid that were sensi- tive to cysteine mutation tended to be clustered onto specific helical faces, suggesting that these faces may be play a more predominant role in the formation of helix-helix contacts. In particular, opposing faces on helix 2 broadly centered around the positions of Met 60 and Gly 62 (Fig. 9A), and the internal face of helix 1 (20) were all mutationally sensitive. The glycine-rich face of helix 3 (positions 101, 104, and 108), which was also acutely sensitive to mutation, is diametrically opposite another sensitive helix 3 region, centered around the position of Ala 106 . It is reasonable to suppose that these faces, along ...
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... of helix 4 with respect to helix 3 is suggested by the suppressor/cross-linking cysteine pair at po- sitions 148 and 103. Orientation of helix 4 such that mutation- ally insensitive sites are excluded from helical contacts, while maintaining proximity of Ile 148 to Ser 103 , places sensitive sites adjacent to the core of the four-helical bundle (Fig. ...
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... assign- ment of the positions of helices 2-4 on the basis of the func- tional effects alone, and several models of helix organization could accommodate the data. However, additional constraints introduced in line with the helix 1-helix 2 cross-linking (Fig. 4) and suppressor mutation data (Table I) leave only one model as the best fit to the data (Fig. 9A). In addition to making an intramolecular contact with helix 1, the cross-linking data dic- tate that helix 2 must also be reasonably close to helix 1 of a neighboring monomer. Specifically, a cysteine at position 25 on helix 1 must be able to make contact with cysteines at positions 56 and 60 on helix 2 of the adjacent polypeptide ...
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... data (Fig. 9A). In addition to making an intramolecular contact with helix 1, the cross-linking data dic- tate that helix 2 must also be reasonably close to helix 1 of a neighboring monomer. Specifically, a cysteine at position 25 on helix 1 must be able to make contact with cysteines at positions 56 and 60 on helix 2 of the adjacent polypeptide (Fig. 9B). Similarly, a cysteine at position 27 must be able to contact position 60 on helix 2. These constraints can be accommodated in the experimental model of the proteolipid, although with relatively large distances between the side chains of these residues. These distances account for the low yield of dimer in helix 1-helix 2 cross-linking ...
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... between the res- idues forming each suppressor pair. Changes in mass of the side chain of the suppressing residue apparently compensate for local disturbances in side chain packing caused by the initial, inhibitory mutation (38). Although these compensatory effects do not necessarily have to be short range, the model of helix organization in Fig. 9 does accommodate the suppressor mutations identified in this study. Suppressor pairs at posi- tions 25/56 and 27/60 reflect the proximity between these res- idues demonstrated by disulfide cross-linking. In addition, the experimental model is consistent with proximity between res- idues forming apparent suppressor pairs between helix 2 ...
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... idues demonstrated by disulfide cross-linking. In addition, the experimental model is consistent with proximity between res- idues forming apparent suppressor pairs between helix 2 and helix 4 at positions 60/137, 59/141, and 60/141. According to the experimental model, all four residues would pack close to the core of the four-helical bundle (Fig. 9B). A previous study using random mutagenesis of the endogenous yeast Vma3p proteo- lipid also identified suppressor mutations at positions equiva- lent to 74/137, 60/141, and 104/137 in the Nephrops proteolipid (39), interpreted as indicating intramolecular contacts between these residues. Such contacts would be consistent with the model ...
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... residues accessible from the lipid phase. Using this approach, we have demonstrated lipid accessibility not only of the wild type Glu 140 , but also of gluta- mate residues introduced mutagenically at positions 64 and 107 on helices 2 and 3, respectively. 2 Proximity of all three residues to the lipid phase is predicted from the model in Fig. ...
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... organization described in Fig. 9 shows some notable parallels with a recently described model of the subunit c oligomer in the F 1 F 0 -ATPase (41,42). This model shows sub- unit c forming a dodecameric ring, with helix 1 oriented toward the center of the complex. Helix 2, which contains the DCCD- reactive Asp 61 , is at the periphery of the complex, forming the ...
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... positions of helices in the model (Fig. 9) were fixed in accordance with the experimental constraints, such that lipid- exposed residues were external to the complex and distances between residues that form strong helix 2/helix 3 intermolecu- lar cross-links were minimized. The helix 1-helix 1 contact that ensues from this helical packing produces an intermolecular distance of ...
Citations
Purpose
The effect of dynamic capabilities on operational excellence and the moderating effect of environmental dynamism on the relationship between operational excellence and dynamic capabilities in the apparel industry in Sri Lanka were investigated while developing new psychometric scales to assess operational excellence and dynamic capacities constructs.
Design/methodology/approach
We followed the exploratory sequential research design with a mixed-method research approach, aligning with the pragmatic research philosophy. Thus, both qualitative and quantitative research methods were followed.
Findings
Dynamic capabilities positively affect operational excellence, and environmental dynamism moderates the relationship between operational excellence and dynamic capabilities in the apparel industry in Sri Lanka such that when a higher environmental dynamism exists, a weaker positive relationship exists between dynamic capabilities and operational excellence. The two main dimensions of the operational excellence construct are continuous improvement of sustainable operational performance and sustainable competitive advantages. It empirically confirmed that sensing, seizing and reconfiguring capabilities are the three main dimensions of the dynamic capabilities construct.
Research limitations/implications
This study was limited to the apparel industry in Sri Lanka. This research phenomenon should be explored in other industrial sectors worldwide to generalize the findings. The practitioners in the apparel sector may improve the organizational dynamic capabilities to achieve operational excellence and keep a strong positive relationship between dynamic capabilities and operational excellence in a highly dynamic environment if they address out-of-family situations with out-of-the-box thinking.
Originality/value
We generated two new empirical findings: (1) dynamic capabilities positively affect operational excellence, and (2) environmental dynamism moderates the relationship between dynamic capabilities and operational excellence. Also, we introduced validated new scales for assessing operational excellence and dynamic capabilities.
Ductins are a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices. The active forms of the Ductins are membranous ring- or star-shaped oligomeric assemblies and they provide various pore, channel, gap-junction functions, assist in membrane fusion processes and also serve as the rotor c-ring domain of V-and F-ATPases. All functions of the Ductins have been reported to be sensitive to the presence of certain divalent metal cations (Me2+), most frequently Cu2+ or Ca2+ ions, for most of the better known members of the family, and the mechanism of this effect is not yet known. Given that we have earlier found a prominent Me2+ binding site in a well-characterised Ductin protein, we hypothesise that certain divalent cations can structurally modulate the various functions of Ductin assemblies via affecting their stability by reversible non-covalent binding to them. A fine control of the stability of the assembly ranging from separated monomers through a loosely/weakly to tightly/strongly assembled ring might render precise regulation of Ductin functions possible. The putative role of direct binding of Me2+ to the c-ring subunit of active ATP hydrolase in autophagy and the mechanism of Ca2+-dependent formation of the mitochondrial permeability transition pore are also discussed.
The vacuolar ATPase has been implicated in a variety of physiological processes in eukaryotic cells. Bafilomycin and concanamycin, highly potent and specific inhibitors of the vacuolar ATPase, have been widely used to investigate the enzyme. Derivatives have been developed as possible therapeutic drugs. We have used random mutagenesis and site-directed mutagenesis to identify 23 residues in the c subunit involved in binding these drugs. We generated a model for the structure of the ring of c subunits in Neurospora crassa by using data from the crystal structure of the homologous subunits of the bacterium Enterococcus hirae (Murata, T., Yamato, I., Kakinuma, Y., Leslie, A. G., and Walker, J. E. (2005) Science 308, 654-659). In the model 10 of the 11 mutation sites that confer the highest degree of resistance are closely clustered. They form a putative drug-binding pocket at the interface between helices 1 and 2 on one c subunit and helix 4 of the adjacent c subunit. The excellent fit of the N. crassa sequence to the E. hirae structure and the degree to which the structural model predicts the clustering of these residues suggest that the folding of the bacterial and eukaryotic polypeptides is very similar.
Apoptosis plays a pivotal role to arbitrate cell deletion in tissue homeostasis, embryological development, and immunological functioning significantly affecting the pathogenesis of different pathological conditions. Neurodegenerative diseases have enormous involvement of deregulated apoptosis. Evidences suggest that chronic neurological disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease and frontotemporal dementia, and a variety of degenerative movement disorders including Parkinson's disease, Huntington's disease adopt apoptosis as major form cell death. Cellular degradation in apoptosis relies on many interlinked signaling cascades. Cellular phenotypes and different offensive causes trigger signaling events, which lead to variations in the morphology of nuclear degradation, particularly those for DNA fragmentation or cell nucleus condensation. Consequently, therapeutics directed toward the management of apoptosis is of significant importance in these diseases. Various strategies have been used to target the precise regulator of apoptosis in neurological such as antisense technology, gene therapy, recombinant biological, classical organic, and combinatorial chemistry. This chapter focuses on different neurological conditions involving apoptosis as a major form of cell death mechanism, the pathophysiological pathways involved, and the future therapies in the disease management.
The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential
for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic
target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing
lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular
isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein
that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across
a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits
c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential.
It also provides the first evidence for the position of subunit e within the complex.
Proton translocation by the vacuolar H(+)-ATPase is mediated by a multicopy transmembrane protein, the 16-kDa proteolipid. It is proposed to assemble in the membrane as a hexameric complex, with each polypeptide comprising four transmembrane helices. The fourth helix of the proteolipid contains an intramembrane acidic residue (Glu140) which is essential for proton translocation and is reactive toward N,N'-dicyclohexylcarbodiimide (DCCD). Current theoretical models of proton translocation by the vacuolar ATPase require that Glu140 should be protonated and in contact with the membrane lipid. In this study we present direct support for this hypothesis. Modification with the fluorescent DCCD analogue N-(1-pyrenyl)cyclohexylcarbodiimide, coupled to fluorescence quenching studies and bilayer depth measurements using the parallax method, was used to probe the position of Glu140 with respect to the bilayer. Glutamate residues were also introduced mutagenically as targets for the fluorescent probe in order to map additional lipid-accessible sites on the 16-kDa proteolipid. These data are consistent with a structural model of the 16-kDa proteolipid oligomer in which the key functional residue Glu140 and discrete faces of the second and third transmembrane helices of the 16-kDa proteolipid are exposed at the lipid-protein interface.
The multicopy subunit c of the H+-transporting F1Fo ATP synthase of Escherichia coli folds across the membrane as a hairpin of two hydrophobic α helices. The subunits interact in a front-to-back fashion, forming
an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp61 in E. coli, centered in the second transmembrane helix is essential for H+ transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28 in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to
the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+pumping for cytoplasmic pH regulation. In this study, Ile28was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled
H+ pumping and passive H+ transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic
pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G
and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.
Papillomaviruses contain a gene, E5, that encodes a short hydrophobic polypeptide that has transforming activity. E5 proteins bind to the 16 kDa subunit c (proteolipid) of the eukaryotic vacuolar H(+)-ATPase (V-ATPase) and this binding is thought to disturb the V-ATPase and to be part of transformation. This link has been examined in the yeast Saccharomyces cerevisiae. The E5 proteins from human papillomavirus (HPV) type 16, bovine papillomavirus (BPV) type 1, BPV-4 E5 and various mutants of E5 and the p12' polypeptide from human T-lymphotropic virus (HTLV) type I all bound to the S. cerevisiae subunit c (Vma3p) and could be found in vacuolar membranes. However, none affected the activity of the V-ATPase. In contrast, a dominant-negative mutant of Vma3p (E137G) inactivated the enzyme and gave the characteristic VMA phenotype. A hybrid V-ATPase containing a subunit c from Norway lobster also showed no disruption. Sedimentation showed that HPV-16 E5 was not part of the active V-ATPase. It is concluded that the binding of E5 and E5-related proteins to subunit c does not affect V-ATPase activity or function and it is proposed that the binding may be due to a chaperone function of subunit c.
The vacuolar H(+)-ATPase provides a channel through which protons can be pumped across the bilayer. It is a complex assembly of about 20 subunits made up from 13 different polypeptide chains. The proton channel is located in the bilayer and therefore must be formed from one or both of the two intramembraneous subunits, called in yeast Vphlp (100 kDa) and Vma3p (16 kDa). Electron microscopy and the use of water soluble and hydrophobic chemical probes in conjunction with mutagenesis to cysteine or glutamic acid residues, suggest that the membrane sector consists of a single Vphlp subunit in association with a hexameric complex of the four-helical bundle Vma3p subunit. This hexamer encloses a large central pore lined by the first transmembrane helix. This pore appears to be impermeable, however; instead, a glutamic acid residue critical to transport function is located on the outside of the hexamer, deeply buried in the membrane and accessible to probes and inhibitors resident in the hydrophobic phase of the bilayer. The stoichiometry and chemistry of inhibitor binding supports the postulate that the mechanism of action involves rotation of the hexamer in the plane of the bilayer. Mutagenesis data on the Vphlp subunit indicate that it is vital to proton transport. It is likely, therefore, that the proton channel is formed at the interface of the Vphlp and Vma3p subunits, the protons moving via a network of interacting charged amino acid side-chains.
The proton-translocating core of eukaryotic vacuolar H(+)-ATPase (V-ATPase), V(0) consists of a hexameric arrangement of transmembrane alpha-helices formed from the related polypeptides, subunit c and subunit c". The former is comprised of four transmembrane alpha-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V(0) contains a minor subunit c-related polypeptide, subunit c'. All three are required for activity of the proton pump in Saccharomyces cerevisiae. We have introduced cysteine residues in the N-terminal extension of subunit c" in a cysteine-free form. All mutant forms are active in the V-ATPase from S. cerevisiae. Oxidation of vacuolar membranes containing the cysteine-replaced forms gave a cross-linked product of 42000Da. Analysis of this species showed it to be a dimeric form of subunit c", and further studies confirmed there are two copies of subunit c" in the V-ATPases in which it is present. Co-expression of double cysteine-replaced forms of both subunit c and c" gave rise to only homotypic cross-linked forms. Also, subunit c oligomeric complexes are present in vacuolar membranes in the absence of subunit c", consistent with previous observations showing hexameric arrangements of subunit c in gap-junction-like membranes. In vitro studies showed subunit c" can bind to subunit c and itself. The extent of binding can be increased by removal of the N-terminal domain of subunit c". This domain may therefore function to limit the copy number of subunit c" in V(0). A deletion study shows that the domain is essential for the activity of subunit c". The results can be combined into a model of V(0) which contains two subunit c" protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V(0) in which subunit c" is present is subunit c(3):subunit c'(1):subunit c"(2). On the basis of the mutational and binding studies, it seems likely that two copies of subunit c" are next to each other.
