Binding assays of inhibitors towards selected V-ATPase domains

The macrolide antibiotic bafilomycin and the related synthetic compound SB 242784 are potent inhibitors of the vacuolar H⁺-ATPases (V-ATPase). It is currently believed that the site of action of these inhibitors is located on the membrane bound c-subunits of V-ATPases. To address the identification of the critical inhibitors binding domain, their specific binding to a synthetic peptide corresponding to the putative 4th transmembrane segment of the c-subunit was investigated using fluorescence resonance energy transfer (FRET), and for this purpose a specific formalism was derived. Another peptide of the corresponding domain of the c′ isoform, was checked for binding of bafilomycin, since it is not clear if V-ATPase inhibition can also be achieved by interaction of the inhibitor with the c′-subunit. It was concluded that bafilomycin binds to the selected peptides, whereas SB 242784 was unable to interact, and in addition for bafilomycin, its interaction with the peptides either corresponding to the c- or the c′-subunit isoforms is identical. Since the observed interactions are however much weaker as compared to the very efficient binding of both bafilomycin and SB 242784 to the whole protein, it can be concluded that assembly of all V-ATPase transmembrane segments is required for an efficient interaction.     

Membrane-bound peptides from V-ATPase subunit a do not interact with an indole-type inhibitor.

The V-ATPases are ATP-dependent proton pumps, found in virtually all cells, responsible for acidification of organelles and energizing of plasma membranes. Its role in diseases, such as osteoporosis and metastatic cancer, makes the V-ATPase a potential drug target. Short synthetic peptides that are presented here mimic the 7th transmembrane domain (TM7) of subunit a (Vph1p) of Saccharomyces cerevisiae V-ATPase, an essential part of the membrane-bound VO domain, where proton translocation takes place. The peptides adopt a transmembrane configuration only in membranes containing anionic lipids, stressing the importance of strong interfacial anchoring by the flanking lysines. Peptide P1, which contains the essential arginine R735, is monomeric, whereas peptide P2, which lacks this extra charge, tends to aggregate in the membrane. SB 242784, which is a highly potent inhibitor of V-ATPase, does not show any interaction with the peptides, indicating that TM7 alone is not sufficient for inhibitor binding.     

Interaction of the indole class of vacuolar H(+)-ATPase inhibitors with lipid bilayers

The selective inhibitor of osteoclastic V-ATPase (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pentadienamide (SB 242784), member of the indole class of V-ATPase inhibitors, is expected to target the membrane-bound domain of the enzyme. A structural study of the interaction of this inhibitor with the lipidic environment is an essential step in the understanding of the mechanism of inhibition. In this work, a comprehensive study of the relevant features of this interaction was performed. Inhibitor partition coefficients to lipid vesicles as well as its transverse location, orientation (order parameters), and dynamics while bound to bilayers were determined through photophysical techniques, taking advantage of the intrinsic fluorescence of the molecule. To better evaluate the functionally relevant features of SB 242784, a second inhibitor, INH-1, from the same class and having a reduced activity was also examined. It is shown that regarding membrane interaction their properties remain very similar for both molecules, suggesting that the differences in inhibition efficiencies are solely a consequence of the molecular recognition processes within the inhibition site in the V-ATPase.     

Subunit a of the yeast V-ATPase participates in binding of bafilomycin

Bafilomycin and concanamycin are potent and highly specific inhibitors of the vacuolar (H(+))-ATPases (V-ATPases), typically inhibiting at nanomolar concentrations. Previous studies have shown that subunit c of the integral V(0) domain participates in bafilomycin binding, and that this site resembles the oligomycin binding site of the F-ATPase (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). Because mutations in F-ATPase subunit a also confer resistance to oligomycin, we investigated whether the a subunit of the V-ATPase might participate in binding bafilomycin. Twenty-eight subunit a mutations were constructed just N-terminal to the critical Arg(735) residue in transmembrane 7 required for proton transport, a region similar to that shown to participate in oligomycin binding by the F-ATPase. The mutants appeared to assemble normally and all but two showed normal growth at pH 7.5, whereas all but three had at least 25% of wild-type levels of proton transport and ATPase activity. Of the functional mutants, three displayed K(i) values for bafilomycin significantly different from wild-type (0.22 +/- 0.03 nm). These included E721K (K(i) 0.38 +/- 0.03 nm), L724A (0.40 +/- 0.02 nm), and N725F (0.54 +/- 0.06 nm). Only the N725F mutation displayed a K(i) for concanamycin (0.84 +/- 0.04 nm) that was slightly higher than wild-type (0.60 +/- 0.07 nm). These results suggest that subunit a of V-ATPase participates along with subunit c in binding bafilomycin.     

Effect of low-molecular peptides on the transformation of fibrinogen into fibrin

The influence of synthetic peptides on fibrinogen transformation to fibrin under the action of thrombin and fibrin-monomer polymerization was investigated. Peptides Gly-Pro-Arg-Pro; Gly-Pro-Arg-Pro-Lys; Gly-Pro-Arg-Pro-Lys-Boc; Gly-Pro-Arg-Pro-Arg are specific inhibitors of fibrin formation. These peptides interfere with the hydrolysing effect of thrombin due to binding to the central domain of fibrinogen. The interaction of peptides with peripheral D-domains of fibrin-monomer may account for polymerization inhibition. The latter peptide has the largest anticoagulation activity. It is likely that arginine in the fifth position stabilizes the structure of the peptides, with the additional epsilon NH2-group activating its interaction with protein.     

Synthetic peptides mimic the assembly of transmembrane glycoproteins

The composition of the intramembranous domains of many receptors are remarkably uniform, yet there is evidence that many transmembrane proteins associate together to form specific noncovalent homo- or heterocomplexes within the membrane. We have synthesized peptides corresponding to transmembrane domains of glycophorin A, glycophorin C, and the interleukin 2-receptor Tac antigen to study the interactions between transmembrane domains in vitro. Synthetic transmembrane glycophorin A peptide formed a complex with native glycophorin and glycoproteins of erythrocyte and K562 cell membranes that was reversible, specific, and could be demonstrated in a natural bilayer system in the absence of detergents. Synthetic glycophorin C and interleukin 2-receptor Tac antigen transmembrane peptides, although similar in amino acid composition, did not interact with glycophorin and did not inhibit the binding of the synthetic glycophorin A transmembrane peptide to native glycophorin. It is proposed that the transmembrane segments of receptor proteins contain not only the structural information necessary for insertion and anchoring but specific binding sites that mediate interactions between transmembrane glycoproteins.     

Conformational studies of peptides representing a segment of TM7 from H+-VO-ATPase in SDS micelles

The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an α-helical conformation or aggregated β-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.e., MTM7). It is concluded that the bulky, positively charged arginine residue located in the center of both peptides has a destabilizing effect on the helical conformation of the SDS-solubilized peptides, leading to β-sheet formation and subsequent aggregation.     

Structural determinants of the factor IX molecule mediating interaction with the endothelial cell binding site are distinct from those involved in phospholipid binding

Previous studies have indicated that Factor IX/IXa interacts in a specific and high affinity manner with a binding site on the endothelial cell surface. In this study, the contributions of the gamma-carboxyglutamic acid-containing (GLA) and growth factor domains to the finding of Factor IX to the endothelium were assessed. While GLA-containing peptides from Factors IX, X, and prothrombin were inhibitors of 125I-Factor IX-endothelial cell binding, the GLA peptide from Factor IX was about 250-800-fold more effective than those from prothrombin and Factor X, respectively. In contrast to its relative efficacy as an inhibitor of Factor IX-cell surface interaction, the Factor IX-GLA peptide neither bound to lipid vesicles nor inhibited Factor IX-lipid interaction. A synthetic peptide comprising the entire first epidermal growth factor (EGF) exon was also an inhibitor of 125I-Factor IX-endothelial cell binding, although it did not interact with lipid vesicles. Experiments with synthetic peptides comprising each of the three loops of the first EGF domain or the entire first EGF region with specific substitutions indicated the importance of determinants in both the first and probably third loops for Factor IX-endothelial interaction. In contrast, the second loop of the first EGF domain and the first loop of the second EGF exon are probably not involved in Factor IX-endothelial interaction based on their inability to block 125I-Factor IX binding to cells. These results indicate that determinants in both the GLA and the first EGF domain contribute to the specific binding of Factor IX to the endothelial cell surface and that structural requirements for Factor IX-cell surface interaction are distinct from those for Factor IX binding to lipids.     

A synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of Sendai virus-cell fusion: an emerging similarity with functional domains of other viruses.

A series of peptides derived from three domains within the fusion protein of Sendai virus was synthesized and examined for their potential to inhibit the fusion of the virus with human red blood cells. These domains include the 'fusion peptide' and two heptad repeats, one adjacent to the fusion peptide (SV-163) and the other to the transmembrane domain (SV-473). Of all the peptides tested, only SV-473 was highly inhibitive. Using fluorescently-labelled peptides, the mechanism through which the SV-473 peptide inhibits the haemolytic activity of the virus was investigated. The results suggest that interactions of the active peptide with virion elements and lipid membranes are involved. Since it has recently been found that synthetic peptides corresponding to putative coiled-coil domains of the human immunodeficiency virus (HIV) type 1 transmembrane protein gp41 are potent inhibitors of HIV, we discuss the general property of virus-derived coiled-coil peptides as inhibitors of viral infection.     

Identification of two domains which mediate the binding of activating phospholipids to the plasma-membrane Ca2+ pump.

The stimulation of the purified human erythrocyte calcium pump by acidic phospholipids was investigated using synthetic peptides corresponding to a putative phospholipid-responsive domain [Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N. & Carafoli, E. (1990) Biochemistry 29, 8070-8076] and to the calmodulin-binding domain of the pump. The peptides interfered with the activation of the enzyme by phosphatidylserine and phosphatidic acid in competition assays. The peptide corresponding to the calmodulin-binding domain was found to be the most efficient antagonist. Direct binding measurements using fluorescent derivatives of the peptides confirmed the interaction between the acidic phospholipids and the peptides, and fluorescence titrations of dansylated calmodulin with the purified ATPase showed a direct effect of acidic phospholipids on calmodulin binding. A proteolyzed preparation of the Ca(2+)-ATPase lacking the calmodulin-binding domain confirmed that the phospholipid-induced stimulation is mediated by two sites, one located in the C-terminal portion of the previously identified 44-amino-acid phospholipid-responsive domain, the other in the calmodulin-binding domain.     

Salicylihalamide A inhibits the V0 sector of the V-ATPase through a mechanism distinct from bafilomycin A1

The newly identified specific V-ATPase inhibitor, salicylihalamide A, is distinct from any previously identified V-ATPase inhibitors in that it inhibits only mammalian V-ATPases, but not those from yeast or other fungi (Boyd, M. R., Farina, C., Belfiore, P., Gagliardi, S., Kim, J. W., Hayakawa, Y., Beutler, J. A., McKee, T. C., Bowman, B. J., and Bowman, E. J. (2001) J. Pharmacol. Exp. Ther. 297, 114-120). In addition, salicylihalamide A does not compete with concanamycin or bafilomycin for binding to V-ATPase, indicating that it has a different binding site from those classic V-ATPase inhibitors (Huss, M., Ingenhorst, G., Konig, S., Gassel, M., Drose, S., Zeeck, A., Altendorf, K., and Wieczorek, H. (2002) J. Biol. Chem. 277, 40544-40548). By using purified bovine brain V-pump and its dissociated V(1) and V(0) sectors, we identified the recognition and binding site for salicylihalamide to be within the V(0) domain. Salicylihalamide does not inhibit the ATP hydrolysis activity of the dissociated V(1)-ATPase but inhibits the ATPase activity of the holoenzyme by inhibiting the V(0) domain. Salicylihalamide causes a dramatic redistribution of cytosolic V(1) from soluble to membrane-associated form, a change not observed in cells treated with either bafilomycin or NH(4)Cl. By synthesizing and characterizing a series of salicylihalamide derivatives, we investigated the structural determinants of salicylihalamide inhibition in terms of potency and reversibility, and used this information to suggest a possible binding mechanism.     

Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM

The neural cell adhesion molecule NCAM is capable of mediating cell-cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoclonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM-covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell-cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP-epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction.     

Peptide fragments of the fractalkine chemokine domain: Influence on migration of human monocytes

The CX3CL1 Fractalkine is the sole cytokine of the CX3C family. Its molecule consists of an extracellular N-terminal chemokine domain, a mucin-like rod, a transmembrane domain, and an intracellular domain. Fractalkine exhibits the properties of an adhesion molecule in the membrane-bound state. The fractalkine chemokine domain (FCD) is proteolytically released from a cellular membrane in a soluble form. It acts as a chemoattractant for leukocytes which express the CX3CR1 fractalkine receptor. Fractalkine participates in the development of a number of pathological inflammation-mediated processes. Therefore, a search for its inhibitors is an urgent problem. We determined the FCD antigenic determinants and synthesized the corresponding peptides: P41-52 H-Leu-Glu-Thr-Arg-Gln-His-Arg-Leu-Phe-Cys-Ala-Asp-NH₂, P53-60 H-Pro-Lys-Glu-Gln-Trp-Val-Lys-Asp-NH₂, and P60-71 H-Asp-Ala-Met-Gln-His-Leu-Asp-Arg-Gln-Ala-Ala-Ala-NH₂. The biological activity of these peptides was evaluated according to their action on the adhesion and migration of human peripheral blood monocytes which expressed the fractalkine receptor. FCD and the P41-52 peptide significantly increased monocyte adhesion and migration in comparison with the corresponding spontaneous adhesion and migration of the cells. The P53-60 and P60-71 peptides inhibited the FCD-stimulated monocyte adhesion and migration. We analyzed the influence of the prepared peptides on the interaction of FCD with heparin by EIA, because binding of chemokines to glycosaminoglycans of cellular surface and extracellular matrix was one of the conditions of the chemokine migration activity. The P41?C52 peptide competed with FCD for the heparin binding, whereas the P53?C60 and P60?C71 peptides had no significant effect.     

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment.     

Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations.

Synthetic peptides corresponding to sequences in the human liver gap junction protein were chemically synthesized and used for generation of peptide antisera to defined sequences in the protein. The antibodies were affinity purified and characterized by demonstrating that they specifically recognized both their corresponding synthetic peptide (as indicated by dot blot analysis) and the native 32-kd gap junction protein (by immunoblotting). The specificity of a subset of the different site-specific antibodies was subsequently confirmed by demonstration of their binding to specific gap junction fragments produced by treatment with a lysine-specific endoproteinase. Immunoelectron microscopy was used to localize the specific peptide antibody epitopes to either the cytoplasmic or extracellular surfaces of the gap junction. Results indicate a transmembrane orientation for the protein with the amino and carboxyl termini located on the cytoplasmic side of the membrane. Based on these data, a model is proposed for the transmembrane folding of the gap junction protein.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Segment TM7 from the cytoplasmic hemi-channel from VO-H+-V-ATPase includes a flexible region that has a potential role in proton translocation

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY (1)H-(1)H NMR spectra are obtained of sMTM7 dissolved in d(6)-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two alpha-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.     

Decreased in vitro fertilization efficiencies in the presence of specific cyritestin peptides

Synthetic peptides corresponding to specific regions of the mouse acrosomal transmembrane protein cyritestin have been used as competitors in in vitro fertilization experiments. One peptide representing a putative egg-receptor binding site within the disintegrin domain of cyritestin lowered the fertilization rate to 30% of the normal value.     

Heptad repeat 2-based peptides inhibit avian sarcoma and leukosis virus subgroup a infection and identify a fusion intermediate

Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.     

Concanamycin and indolyl pentadieneamide inhibitors of the vacuolar H+-ATPase bind with high affinity to the purified proteolipid subunit of the membrane domain

The macrolide antibiotic concanamycin is a potent and specific inhibitor of the vacuolar H(+)-ATPase (V-ATPase), binding to the V(0) membrane domain of this eukaryotic acid pump. Although binding is known to involve the 16 kDa proteolipid subunit, contributions from other V(0) subunits are possible that could account for the apparently different inhibitor sensitivities of pump isoforms in vertebrate cells. In this study, we used a fluorescence quenching assay to directly examine the roles of V(0) subunits in inhibitor binding. Pyrene-labeled V(0) domains were affinity purified from Saccharomyces vacuolar membranes, and the 16 kDa proteolipid was subsequently extracted into chloroform and methanol and purified by size exclusion chromatography. Fluorescence from the isolated proteins was strongly quenched by nanomolar concentrations of both concanamycin and an indolyl pentadieneamide compound, indicating high-affinity binding of both natural macrolide and synthetic inhibitors. Competition studies showed that these inhibitors bind to overlapping sites on the proteolipid. Significantly, the 16 kDa proteolipid in isolation was able to bind inhibitors as strongly as V(0) did. In contrast, proteolipids carrying mutations that confer resistance to both inhibitors showed no binding. We conclude that the extracted 16 kDa proteolipid retains sufficient fold to form a high-affinity inhibitor binding site for both natural and synthetic V-ATPase inhibitors and that the proteolipid contains the major proportion of the structural determinants for inhibitor binding. The role of membrane domain subunit a in concanamycin binding and therefore in defining the inhibitor binding properties of tissue-specific V-ATPases is critically re-assessed in light of these data.