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

African Viper Poly-His Tag Peptide Fragment Efficiently Binds Metal Ions and Is Folded into an α‑Helical Structure

Snake venoms are complex mixtures of toxic and often spectacularly biologically active components. Some African vipers contain polyhistidine and polyglycine peptides, which play a crucial role in the interaction with metal ions during the inhibition of snake metalloproteases. Polyhistidine peptide fragments, known as poly-His tags, play many important functions, e.g., in metal ion transport in bacterial chaperon proteins. In this paper, we report a detailed characterization of Cu²⁺, Ni²⁺, and Zn²⁺ complexes with the EDDHHHHHHHHHG peptide fragment (pHG) derived from the venom of the rough scale bush viper (Atheris squamigera). In order to determine the thermodynamic properties, stoichiometry, binding sites, and structures of the metal–pHG complexes, we used a combination of experimental techniques (potentiometric titrations, electrospray ionization mass spectrometry, UV–vis spectroscopy, circular dichroism spectroscopy, and electron paramagnetic resonance spectroscopy) and extensive computational tools (molecular dynamics simulations and density functional theory calculations). The results showed that pHG has a high affinity toward metal ions. The numerous histidine residues located along this sequence are efficient metal ion chelators with high affinities toward Cu²⁺, Ni²⁺, and Zn²⁺ ions. The formation of an α-helical structure induced by metal ion coordination and the occurrence of polymorphic binding states were observed. It is proposed that metal ions can “move along” the poly-His tag, which serves as a metal ion transport pathway. The coordination of Cu²⁺, Ni²⁺, and Zn²⁺ ions to the histidine tag is very effective in comparison with other histidine-rich peptides. The stabilities of the metal–pHG complexes increase in the order Zn²⁺ < Ni²⁺≪ Cu²⁺