Specificity of the Zn2+, Cd2+ and Ni2+ ion binding sites in the loop domain of the HypA protein

The zinc binding loop domain of the HypA protein of Helicobacter pylori consists of two CXXC motifs with flanking His residues. These motifs bind metal ions, and thus they are crucial for the functioning of the whole protein. The N-terminal site, where His is separated from CXXC by Ser residue is more effective in binding Zn2+ and Ni2+ ions than the C-terminal site, in which His is adjacent to the CXXC motif. Studies on various modifications of the peptide sequence within the Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 loop show the role of the residues in the linker between the CXXC motifs and the effect of the length of the linker on the stability of the complexes it forms with Zn2+, Cd2+ and Ni2+ ions. The proline residue in the linker between the two CXXC binding sites plays a distinct role in the metal ion binding ability of the loop, lowering the efficacy of the metal ion coordination. The deletion of the aliphatic residues from the linker between the CXXC motifs remarkably improves the binding efficacy of the loop. © The Royal Society of Chemistry

Effect of antisense peptide binding on the dimerization of human cystatin C - gel electrophoresis and molecular modeling studies

Human cystatin C (HCC) shows a tendency to dimerize. This process is particularly easy in the case of the L68Q HCC mutant and might lead to formation of amyloid deposits in brain arteries of young adults. Our purpose was to find ligands of monomeric HCC that can prevent its dimerization. Eleven antisense peptide ligands of monomeric HCC were designed and synthesized. The influence of these ligands on HCC dimerization was studied using gel electrophoresis and molecular modeling methods. The results suggest that all the designed peptides interact with monomeric HCC facilitating its dimerization rather than preventing it

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?

Human L68Q cystatin C is one of the known human amyloidogenic proteins. In its native state it is a monomer with alpha/beta structure. Experimental evidence suggests that L68Q variant associates into dimeric intermediates and that the dimers subsequently self-assemble to form amyloid deposits and insoluble fibrils. Details of the pathway of L68Q mutant amyloid formation are unclear; however, different experimental approaches with resolutions at molecular level have provided Some clues. Probably, the stability and flexibility of monomeric L68Q variant play essential roles in the early steps of amyloid formation; thus, it is necessary to characterize early conformational changes of L68Q cystatin C monomers. In this paper, we demonstrate the possibility that the differences between the monomeric forms of wild-type (wt) cystatin C and its L68Q variant are responsible for higher tendency of the L68Q cystatin C amyloidogenesis. We started our studies with the simulations of wt and L68Q cystatin C monomers. Nanosecond time scale molecular dynamics simulations at 308 K were performed using AMBER7.0 program, The results show that the structure of the L68Q monomer was changed, relative to the wt cystatin C structure. The results support earlier speculation that the L68Q point mutation would easily lead to dimer formation. (c) 2006 Published by Elsevier Inc

Novel azapeptide inhibitors of cathepsins B and K. Structural background to increased specificity for cathepsin B

We have designed and synthesized a new series of azapeptides which act as potential inhibitors of cathepsin B and/or cathepsin K. Their structures are based upon the inhibitory sites of natural cysteine protease inhibitors, cystatins. For the synthesized azapeptides, the equilibrium constants for dissociation of inhibitor-enzyme complex, K-i, were determined. Comparison of these values indicated that all of the azainhibitors act much stronger toward cathepsin B. Z-Arg-Leu-His-Agly-Ile-Val-OMe (7) proved to be approximately 500 times more potent for cathepsin B than for cathepsin K. To be able to explain the obtained experimental values we used the molecular dynamics procedures to analyze the interactions between cathepsin B and compound 7. We also determined the structure of the most potent and selective cathepsin B azainhibitor by means of NMR studies and theoretical calculations. In this report, we describe SAR studies of azapeptide inhibitors indicating the influence of the conformational flexibility of the examined compounds on inhibition of cathepsins B and K

Cyclic trimer of human cystatin C, an amyloidogenic protein Molecular dynamics and experimental studies

Human cystatin C (HCC) is a cysteine protease inhibitor that takes a series of oligomeric forms in solution (e.g., dimers, trimers, tetramers, decamers, dodecamers, and other higher oligomers). The best-known form of cystatin C is the dimer, which arises as a result of a domain swapping mechanism. The formation of the HCC oligomeric forms, which is most likely due to this domain swapping mechanism, is associated with the aggregation of HCC into amyloid fibrils and deposits. To investigate the structure of a specific HCC oligomer, we developed a covalently stabilized trimer of HCC. An atomic model of this HCC trimer was proposed on the basis of molecular docking and molecular dynamics simulations. The most stable model of the HCC trimer obtained from the molecular dynamics simulations is characterized by a well-preserved secondary structure. The molecular size and structural parameters of the HCC trimer in solution were also confirmed by Small Angle Neutron Scattering and Nuclear Magnetic Resonance Diffusometry

Polyproline II conformation is one of many local conformational states and is not an overall conformation of unfolded peptides and proteins

The alanine-based peptide Ac-XX(A)(7)OO-NH(2), referred to as XAO (where X, A, and O denote diaminobutyric acid, alanine, and ornithine, respectively), has recently been proposed to possess a well defined polyproline II (P(II)) conformation at low temperatures. Based on the results of extensive NMR and CD investigations combined with theoretical calculations, reported here, we present evidence that, on the contrary, this peptide does not have any significant amount of organized P(II) structure but exists in an ensemble of conformations with a distorted bend in the N- and C-terminal regions. The conformational ensemble was obtained by molecular dynamics/simulated annealing calculations using the amber suite of programs with time-averaged distance and dihedral-angle restraints obtained from rotating-frame nuclear Overhauser effect (ROE) volumes and vicinal coupling constants (3)J(HNΗα), respectively. The computed ensemble-averaged radius of gyration R(g) (7.4 ± 1.0) Å is in excellent agreement with that measured by small-angle x-ray scattering (SAXS) whereas, if the XAO peptide were in the P(II) conformation, R(g) would be 11.6 Å. Depending on the pH, peptide concentration, and temperature, the CD spectra of XAO do or do not possess the maximum with positive ellipticity in the 217-nm region, which is characteristic of the P(II) structure, reflecting a shifting conformational equilibrium rather than an all-or-none transition. The “P(II) conformation” should, therefore, be considered as one of the accessible conformational states of individual amino acid residues in peptides and proteins rather than as a structure of most of the chain in the early stage of folding