Zbigniew Grzonka

Exploration of the conformational space of oxytocin and arginine-vasopressin using the electrostatically driven Monte Carlo and molecular dynamics methods

Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine-vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration-shell model, and by simulated-annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low-energy conformations obtained for both hormones were classified using the minimal-tree clustering algorithm and characterized according to the locations of beta-turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a beta-turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a beta-turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The latter difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x-ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr-derived distance constraints. Generally, the low-energy conformations obtained with the hydration-shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low-energy conformations do not satisfy all of the nmr-derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with beta-turns at residues 2,3, 3,4, and 4,5. The low-energy structures of OT with a beta-turn at residues 2,3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M. D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921-927], although the native hormone differs from the bicyclic analogue by the conformation of the C-terminal tripeptide. This finding confirms the hypothesis of different receptor-bound conformations of agonists and antagonists of OT.

Interactions of vasopressin agonists and antagonists with membrane receptors

Plasma membranes containing one class of non-cooperative binding sites for tritium-labelled [8-arginine]vasopressin were isolated from bovine kidney inner medulla and from rat liver. By using a weighted, non-linear least squares fit to logistic curves, the binding parameters of eight vasopressin agonists and antagonists were determined in competition experiments. Vasopressin analogues with sarcosine or N-methyl-L-alanine in position 7 instead of proline showed a high ratio of antidiuretic to vasopressor activity. These analogues retained a high binding affinity to the renal vasopressin receptor with apparent dissociation constants KD in the order proline less than sarcosine less than methylalanine . In contrast, the affinity to the hepatic vasopressin receptor, which shares characteristics with vasopressor receptors, was drastically reduced with KD values being in the order proline much less than N- methylalanine less than sarcosine. By combining the substitutions at position 7 with substitutions of cysteine in position 1 by either deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid, inhibitors of the oxytocoic and vasopressor responses were obtained. These additional substitutions at position 1 led to a drastic decrease in the binding affinity to the vasopressin receptor in bovine kidney. The intrinsic activity of these analogues to stimulate the renal vasopressin sensitive adenylate cyclase was strongly reduced or completely lost. In the rat liver system, however, these vasopressin antagonists showed a remarkably increased affinity to vasopressin receptors as compared to analogues substituted only at position 7. GTP reduced the binding affinity of all analogues to the hepatic receptor. The results show that these structural modifications which influence both the conformational properties of the vasopressin molecule and the biological activities of the hormone had strikingly different effects on the interactions of the resulting analogues with physiologically important receptors in the kidney and the liver. These studies may lead to the development of more specific vasopressin agonists and antagonists.

Binding Epitopes and Interaction Structure of the Neuroprotective Protease Inhibitor Cystatin C with beta-Amyloid Revealed by Proteolytic Excision Mass Spectrometry and Molecular Docking Simulation

Human cystatin C (HCC) is a protease inhibitor with a propensity to form beta-amyloid (Abeta)-like fibrils and to coassociate with amyloidogenic proteins. Recently, a specific interaction between HCC and Abeta has been found. Here, we report the identification of the Abeta and HCC binding epitopes in the Abeta-HCC complex, using a combination of selective proteolytic excision and high resolution mass spectrometry. Proteolytic excision of Abeta(1-40) on sepharose-immobilized HCC and MALDI-MS identified the epitope Abeta(17-28). On immobilized Abeta(1-40), affinity MS of HCC fragments identified a specific C-terminal epitope, HCC(101-117). Binding specificities of both epitopes were ascertained by ELISA and surface plasmon resonance and by direct electrospray MS of the HCC-Abeta epitope peptide complexes. A structure model of the HCC-Abeta complex by molecular docking simulation showed full agreement with the identified Abeta and HCC epitopes. Inhibition studies in vitro revealed Abeta-fibril inhibiting activity of the HCC(101-117)-epitope. The Abeta-HCC interacting epitopes provide lead structures of neuroprotective inhibitors for AD and HCC amyloidosis therapy.

Synthesis of cysteine proteinase inhibitors structurally based on the proteinase interacting N-terminal region of human cystatin C.

Fibronectin contains two latent gelatinolytic enzymes, FN-gelatinase and FN-laminase that can be activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. The results of this work show that Achromobacter collagenase cleaves fibronectin and generates an active FN-gelatinase. In contrast to the cathepsin D digest, the collagenase digest directly exhibits gelatinolytic activity without additional activation. The gelatinolytic activity of the total collagenase digest can be inhibited by phenylmethanesulfonyl fluoride, a serine proteinase inhibitor and by pepstatin A, an aspartic-acid proteinase inhibitor. FN-laminase activity, when assayed with its synthetic substrate GPAGPR and also with laminin was revealed after separation of the collagenase digest of fibronectin on heparin Ultrogel. FN-gelatinase and FN-laminase activities were found in heparin unretained and heparin strongly retained fractions. These results have demonstrated that in contrast to cathepsin D, Achromobacter collagenase activates two matrix-degrading proteinases from fibronectin, FN-Gelatinase und FN-Laminase.

[125I]-[d(CH2)5, Sar7]AVP: a selective radioligand for V1 vasopressin receptors.

Arginine8-vasopressin (AVP) acts on vascular and hepatic V1 receptors to influence blood pressure and glycogenolysis respectively. We have radioiodinated the AVP V1 receptor antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic-acid), 7-sarcosine, 8-arginine] vasopressin ([d(CH2)5, Sar7]AVP) and determined its receptor-binding properties in rat liver and kidney plasma membranes. The binding was of high affinity to single classes of receptors (liver: Kd = 3.0 nM and Bmax = 530 +/- 10 fmol/mg protein, kidney: Kd = 0.5 +/- 0.9 nM and Bmax = 11 +/- 8 fmol/mg protein). Competition of [125I]-[d(CH2)5, Sar7]AVP binding by unlabelled AVP analogues gave the following order of potency in both tissues, consistent with that expected for binding to a V1 receptor: [d(CH2)5, Tyr(Me)2]AVP greater than AVP greater than [d(CH2)5, D-Ile2, Ile4] AVP greater than DDAVP. No degradation of [125I]-[d(CH2)5, Sar7]AVP during incubation or storage was detected by HPLC analysis. We have used [125I]-[d(CH2)5, Sar7]AVP and in vitro autoradiography to demonstrate its use in localizing brain AVP receptors. These studies suggest that [125I]-[d(CH2)5, Sar7]AVP is a suitable selective radioligand for labelling V1 receptors and will provide a valuable tool for the study of the localization and regulation of AVP V1 receptors in tissues and in receptor isolation.

Copper(II) binding by fragments of α-synuclein containing M1-D2- and -H50-residues; a combined potentiometric and spectroscopic study

Stability constants and ligand donor sets of the copper(II) complexes of the NH2-29-56(L1)(AA30GKTKEGVLYV40GSKTKEGVVH50GVATVA56-NH2), NH2-M29-D30-56(L2) and Ac-M29-D30-56(L3) fragments of alpha-synuclein were determined in aqueous solution for 1 : 1 metal-to-ligand molar ratio in the pH range 2.5-10.5. The tyrosine residue in the 39th position of the alpha-synuclein fragments does not take part in the coordination of the metal ion. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the L3 fragment compared to that of the L2 peptide. When the amino group is blocked (L3) the imidazole nitrogen of the histidine residue acts as an anchoring site and at higher pH the 3N {N(Im),2N-} and 4N {N(Im),3N-} complexes are formed. The L1 peptide at physiological pH forms in equilibrium 3N {NH2,N-,CO,N(Im)} and 4N {NH2,2N-,N(Im)} complexes. For the L2 peptide the coordination of the copper(II) ions starts from the N-terminal Met residue and with increasing of pH the Asp residue in second position of amino acid sequence coordinates and stabilizes significantly the 2N complex as a result of chelation through the beta-carboxylate group. At physiological pH the 3N {NH2,N-,beta-COO-,N(Im)} coordination mode dominates. At pH above 6 the results for the L2 fragment suggest the formation of 3N and 4N complexes (in equatorial plane) and the involvement of the lateral NH2 group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra sigma (epsilon-NH2-Lys) --> Cu(II) charge transfer transition is observed. The stability constants for the L2 fragment of alpha-synuclein of the 4N {NH2,2N-,N(Im)} and {NH2,3N-} complexes are higher about 1.5 and 0.7 orders of magnitude, respectively, by comparison to those of the L1 peptide. This increase may be explained by the involvement of the epsilon-NH2 group of Lys residue in the coordination sphere of metal ion.

A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro

Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg(8)-Leu(9)-Val(10)-Gly(11) (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg(8)-Leu(9)-Val(10)-Gly(11)-diazomethane (Z-RLVG-CHN(2)) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of (45)Ca and (3)H from mouse calvarial bones prelabeled with [(45)Ca]CaCl(2) and [(3)H]-proline, respectively. Z-RLVG-CHN(2) concentration-dependently inhibited the release of (45)Ca and (3)H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 micromol/L. The inhibitory actions of Z-RLVG-CHN(2) and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN(2) and cystatin C on PTH-stimulated (45)Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN(2) and cystatin C, as well as that of calcitonin, on (3)H release was seen already after 2 h. Z-RLVG-CHN(2), in which the reactive carboxyterminal diazomethane was substituted by nonreactive groups [-OH, -NH(2), or -N(CH(3))(2)], resulted in peptidyl derivatives, which, in contrast to Z-RLVG-CHN(2) and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg(8), Leu(9), Val(10), and Trp(106), and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN(2) inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.

FTIR spectroscopic studies on aggregation process of the β-amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimers disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 310‐helix/310‐turn structures. Copyright

Interactions of Cu2+ ions with chicken prion tandem repeats.

The potentiometric and spectroscopic (EPR, UV-Vis, CD) data have shown that the chicken prion hexa-repeat (Ac-His-Asn-Pro-Gly-Tyr-Pro-NH(2)) is a very specific ligand for Cu(2+) ions. The His imidazole is an anchoring binding site, then the adjacent amide nitrogen coordinates as a second donor. The presence of Pro at position 3 induces binding of phenolate oxygen as a third donor atom. The tridentate coordination dominates around physiological pH. Similar to human octapeptide fragments, chicken tandem repeats exhibit a cooperative effect in binding Cu(2+) ions, although chicken peptides are much less effective in metal ion coordination.

The whole hexapeptide repeats domain from avian PrP displays untypical hallmarks in aspect of the Cu2+ complexes formation

Prions, the infectious agents responsible for the transmissible spongiform encephalopathies (TSEs) have defied full characterization for decades. Although the interactions of Cu2+ ions with PrP both in vivo and in vitro are well documented, there are still a lot of ambiguities concerning the biological and chemical nature of these effects. In this work, we have investigated the interactions of Cu2+ ions with whole repeat region of the copper‐binding domain (hexapeptide repeats) of chicken PrP. Our results provide explanations for the structural and chemical basis of the specific interactions of Cu2+ ions with the hexapeptide repeat region. Furthermore, we show that SOD‐like activity depends on Cu2+ complexes.