Conformation of dioxopiperazines,II. CNDO/2 quantum mechanical calculations on conformational preferences in cyclo (glycyl-L-alanyl), cyclo(glycyl-L-valyl), and both epimers of cyclo-di-(alanyl)

Conformational energy calculations, aimed at verification of the suitability of the semiempirical molecular orbital CNDO/2 method for conformational elucidations in cyclic dipeptides formed from amino acids with aliphatic side chains, have been carried out. The results obtained for four dioxopiperazines [DOP; cyclo(Glycyl-L-Alanyl), cyclo(Glycyl-L-Valyl), and both epimers of cyclo-di-(Alanyl)] point out very good agreement with experimental premises. The latter include (1) the preference of the cis-peptide bonds for being nonplanar, which results in twisted-boat conformations of the DOP ring; (2) greater stability of conformers with a side chain oriented axially over those with a side chain oriented equatorially; (3) the preference of cyclo(Gly-Val) for assuming a folded conformation with one of the side chain γ-methyl groups sticking over the DOP ring.     

Prediction of conformation of rat galanin in the presence and absence of water with the use of monte Carlo methods and the ECEPP/3 force field

The conformation of the 29-residue rat galanin neuropeptide was studied using the Monte Carlo with energy minimization (MCM) and electrostatically driven Monte Carlo (EDMC) methods. According to a previously elaborated procedure, the polypeptide chain was first treated in a united-residue approximation, in order to enable extensive exploration of the conformational space to be carried out (with the use of MCM), Then the low-energy united-residue conformations were converted to the all-atom representations, and EDMC simulations were carried out for the all-atom polypeptide chains, using the ECEPP/3 force field with hydration included. In order to estimate the effect of environment on galanin conformation, the low-energy conformations obtained as a result of these simulations were taken as starting structures for further EDMC runs that did not include hydration. The lowest-energy conformation obtained in aqueous solution calculations had a nonhelical N-terminal part packed against the nonpolar face of a residual helix that extended from Pro¹³ toward the C-terminus. One next lowest-energy structure was a nearly-all-helical conformation, but with a markedly higher energy. In contrast, all of the low-energy conformations in the absence of water were all-helical differing only by the extent to which the helix was kinked around Pro¹³. These results are in qualitative agreement with the available NMR and CD data of galanin in aqueous and nonaqueous solvents.     

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli: STRUCTURAL AND FUNCTIONAL STUDIES*

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA′ (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.     

Signal transmission via G protein-coupled receptors in the light of rhodopsin structure determination

G protein-coupled receptors (GPCRs) transducing diverse external signals to cells via activation of heterotrimeric GTP-binding (G) proteins, estimated to mediate actions of 60% of drugs, had been resistant to structure determination until summer 2000. The first atomic-resolution experimental structure of a GPCR, that of dark (inactive) rhodopsin, thus provides a trustworthy 3D prototype for antagonist-bound forms of this huge family of proteins. In this work, our former theoretical GPCR models are evaluated against the new experimental template. Subsequently, a working hypothesis regarding the signal transduction mechanism by GPCRs is presented.     

Molecular simulations of rhodopsin tetrameter

Di/oligomerization of G-protein coupled receptors (GPCRs) is well established, however very little is known regarding the interaction details. Current paper presents results of molecular dynamics simulations of theoretical model of rhodopsin tetramer with transducine (Gt) in lipid bilayer. Ligand-protein and receptor-receptor interactions have been proposed.     

Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases.     

Conformation of cyclo-(D-phenylalanyl-trans-4-fluoro-D-prolyl)

cyclo(D-Phenylalanyl-trans-4-fluoro-D-prolyl), c(D-Phe-D-FPro), was synthesized and its conformation determined both in solution and in the solid state by 1H NMR and X-ray analysis, respectively. In the crystals the 2.5-diketopiperazine (DKP) ring assumes the uncommon conformation, for cyclodipeptides containing Pro residue, of a flattened chair, which seemingly results from a compromise between, on the one hand, the DKP-aromatic intramolecular ring-ring attraction (folding), requiring the C alpha--C beta bond of the Phe to be axial, and, on the other hand, the intrinsic tendency of the Pro residue to have its C alpha--C beta bond equatorial. Unlike the solid state, the 1H NMR data in CDCl3 and C6D6 demonstrate that in both solutions the DKP ring assumes a boat-like shape, typical for the Pro-containing cyclodipeptides, with the equatorial C alpha--C beta bonds in both amino acid residues, which preclude ring-ring folding. A similar conformation was encountered in the closest analog of c(D-Phe-D-FPro), viz, in c(Phe-Pro), both in solution (21, 22, 26) and in the solid state (12). A subtle interplay of intramolecular interactions introduced into a cyclodipeptide by a Pro-type and a Phe-type residue is emphasized.     

Analysis of interactions responsible for vasopressin binding to human neurohypophyseal hormone receptors-molecular dynamics study of the activated receptor-vasopressin-G(alpha) systems.

Vasopressin (CYFQNCPRG-NH(2), AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G-protein-coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptors versus the closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three-dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(338-350) prototype as a template. AVP was docked into the receptor-G(alpha) systems. The three lowest-energy pairs of receptor-AVP-G(alpha) (two complexes per each receptor) were selected. The 1-ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER 7.0 force field. Six relaxed receptor-AVP-G(alpha) models were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed.     

Conformational studies of neurohypophyseal hormones analogues with glycoconjugates by NMR spectroscopy

Two glycosylated peptides have been studied using NMR spectroscopy supported by molecular modeling. Peptide I is an oxytocin (OT) analogue in which glutamine 4 was replaced by serine with attached α‐d ‐mannose through the oxygen β atom, whereas peptide II is a lysine‐vasopressin (LVP) analogue with lysine 8 side chain modified by the attachment of glucuronic acid through an amide bond. Both peptides exhibit very weak uterotonic effect and are less susceptible to proteolytic degradation than the mother hormones. Additionally, peptide II reveals very weak pressor and antidiuretic activities. Our results have shown that the conformational preferences of glycosylated analogues are highly similar to those of their respective mother hormones. OT glycosylated analogue (I) exhibits a 3,4 β‐turn characteristic of OT‐like peptides, and vasopressin‐glycosylated analogue (II) exhibits β‐turns typical of vasopressin‐like peptides. Therefore, the lack of binding of the glycosylated analogues to the receptors can be attributed to a steric interference between the carbohydrate moieties and the receptors. We also consider this to be the reason of the very low activity of the analyzed glycopeptides. We expect that results from these studies will be helpful in designing new OT‐like and vasopressin‐like drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.