Binding of a C-end rule peptide to the neuropilin-1 receptor: a molecular modeling approach

Neuropilin-1 (NRP-1) is a receptor that plays an essential role in angiogenesis, vascular permeability, and nervous system development. Previous studies have shown that peptides with an N-terminal Arg, especially peptides with the four-residue consensus sequence R/K/XXR/K, bind to NRP-1 cell surfaces. Peptides containing such consensus sequences promote binding and internalization into cells, while blocking the C-terminal Arg (or Lys) prevents the internalization. In this study, we use molecular dynamics simulations to model the structural properties of the NRP-1 complex with a prototypic CendR peptide, RPAR. Our simulations show that RPAR binds NRP-1 through specific interactions of the RPAR C-terminus: three hydrogen bonds and a salt bridge anchor the ligand in the receptor pocket. The modeling results were used as the starting point for a systematic computational study of new RPAR analogues based on chemical modifications of their natural amino acids. Comparison of the structural properties of the new peptide-receptor complexes with the original organization suggests that some of the analogues can increase the binding affinity while reducing the natural sensitivity of RXXR to endogenous proteases.     

Deltorphin analogs restricted via a urea bridge: structure and opioid activity.

Eight cyclic heptapeptides related to the full sequence of deltorphin have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin. Depending on protection procedures, the N-protected peptide-resins or N-protected peptide amides with free amino groups in the side chains were obtained, which were subsequently treated with bis-(4-nitrophenyl)carbonate to form a urea unit. Opioid activities of the peptides were determined in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Several compounds showed high delta opioid agonist potency and high selectivity for delta receptors. The results were compared with those obtained earlier for respective 1-4 deltorphin analogs. The conformations of these peptides have been studied using 2D-NMR in H2O/D2O and molecular dynamics. We observed that the backbone rings had well defined conformations, while the Tyr and Phe side chains and the C-terminal tail had significant conformational freedom. The bioassay data and conformational parameters of these peptides were compared with those of previously described, corresponding 1-4 deltorphin analogs. This comparison permitted an assessment of the role of the C-terminal peptide segment in defining the conformation and receptor interaction of the N-terminal portion and provided insight into the relationship between the putative bioactive conformations and bioactivity.     

Thrombin receptor-activating peptides (TRAPs): investigation of bioactive conformations via structure-activity, spectroscopic, and computational studies

The thrombin receptor (PAR-1) is an unusual transmembrane G-protein coupled receptor in that it is activated by serine protease cleavage of its extracellular N-terminus to expose an agonist peptide ligand, which is tethered to the receptor itself. Synthetic peptides containing the agonist motif, such as SFLLRN for human PAR-1, are capable of causing full receptor activation. We have probed the possible bioactive conformations of thrombin receptor-activating peptides (TRAPs) by systematic introduction of certain conformational perturbations, involving alpha-methyl, ester psi(COO), and reduced-amide psi(CH2N) scans, into the minimum-essential agonist sequence (SFLLR) to probe the importance of the backbone conformation and amide NH hydrogen bonding. We performed extensive conformational searches of representative pentapeptides to derive families of putative bioactive structures. In addition, we employed 1H NMR and circular dichroism (CD) to characterize the conformational disposition of certain pentapeptide analogues experimentally. Activation of platelet aggregation by our pentapeptide analogues afforded a structure-function correlation for PAR-1 agonist activity. This correlation was assisted by PAR-1 receptor binding data, which gauged the affinity of peptide ligands for the thrombin receptor independent of a functional cellular response derived from receptor activation (i.e. a pure molecular recognition event). Series of alanine-, proline-, and N-methyl-scan peptides were also evaluated for comparison. Along with the known structural features for PAR-1 agonist peptides, our work adds to the understanding of peptide topography relative to platelet functional activity and PAR-1 binding. The absolute requirement of a positively charged N-terminus for strong agonist activity was contradicted by the N-terminal hydroxyl peptide psi(HO)S-FLLR-NH2. The amide nitrogen between residues 1 and 2 was found to be a determinant of receptor recognition and the carbonyl groups along the backbone may be involved in hydrogen bonding with the receptor. Position 3 (P3) of TRAP-5 is known to tolerate a wide variety of side chains, but we also found that the amide nitrogen at this position can be substituted by an oxygen, as in SF-psi(COO)-LLR-NH2, without diminishing activity. However, this peptide bond is sensitive to conformational changes in that SFPLR-NH2 was active, whereas SF-NMeL-LR-NH2 was not. Additionally, we found that position 3 does not tolerate rigid spacers, such as 3-aminocyclohexane-1-carboxylic acid and 2-aminocycloalkane-1-carboxylic acid, as analogues 1A, 1B, 2A, 2B, 3, 4, 5A and 5B lack agonist activity. On the basis of our results, we suggest that an extended structure of the agonist peptide is principally responsible for receptor recognition (i.e. binding) and that hydrophobic contact may occur between the side chains of the second (Phe) and fourth (Leu) residues (i.e. P2-P4 interaction).     

High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.     

Effect of lengthening of peptide backbone by insertion of chiral beta-homo amino acid residues: conformational behavior of linear peptides containing alternating L-leucine and beta-homo L-leucine residues

The synthesis and the solution behavior of the linear peptides containing a beta-homo (beta-H) leucine residue-Boc-Leu-beta-HLeu-Leu-OMe, Boc-beta-HLeu-Leu-beta-HLeu-Leu-OMe, and Boc-Leu-beta-HLeu-Leu-beta-HLeu-Leu-OMe-as well as the solid structure of the tripeptide, are reported. The conformational behavior of the peptides was investigated in solution by two-dimensional nmr. Our data support the existence in solution with different families of conformers in rapid interchange. The crystals of the tripeptide are orthorhombic, space group P2(1)2(1)2, with a = 15.829(1) A, b = 29.659(1) A, c = 6.563(1) A, and Z = 4. The structure has been solved by direct methods and refined to final R1 and wR2 indexes of 0.0530 and 0.1436 for 2420 reflections with I > 2sigma(I). In the solid state, the tripeptide does not present intramolecular H bonds, and the peptide backbone of the two leucine residues adopts a quasi-extended conformation. For the beta-HLeu residue, the backbone conformation is specified by the torsion angles straight phi(2) = -120.9(4) degrees, mu(2) = 56.7(4) degrees, psi(3) = -133.2(4) degrees. The side chains of the three residues assume the same conformation (g(-), g(-), trans), and all peptide bonds, except the urethane group at the N-terminus, are in the trans conformation. Preliminary conformational energy calculations carried out on the Ac-NH-beta-HAla-NHMe underline that the conformations with mu angle equal to 180 degrees and 60 degrees assume lower energy with respect to the others. In addition, we found a larger conformational freedom for the psi angle with respect to the straight phi angle.     

Molecular-dynamics simulations of [Met5]- and [D-Ala2,Met5]-enkephalins. Biological implication of monomeric folded and dimeric unfolded conformations.

To investigate the biologically active conformation of enkephalin, molecular-dynamics simulations were applied to [Met5]- and [D-Ala2,Met5]-enkephalins. The dynamic trajectory of monomeric extended [Met5]-enkephalin was analysed in terms of relative mobility between respective torsions of backbone chain. After 10 ps of the dynamics simulation, the conformational transition was converged into a stationary state among the beta-bend folded forms, where they are stabilized by several intramolecular hydrogen-bond formations. Similar conformational transition was also observed in the dynamics simulation of [D-Ala2,Met5]enkephalin, which is a more mu-receptor-specific peptide than [Met5]enkephalin. The geometrical correspondence between the monomeric enkephalin conformation in the stationary state and morphine molecule (a mu-specific rigid opiate) was surveyed by virtue of the triangular substructures generated by choosing three functional atoms in each molecule, and good resemblances were observed. On the other hand, the dynamics simulation of the antiparallel extended [Met5]enkephalin dimer showed a trajectory different from that of the monomeric one. Two intermolecular hydrogen bonds at Tyr1 (NH3+)...Met5(CO2-) end residues were held throughout the 100 ps simulation, the dimeric structure being consequently kept. The conformational transition of the backbone chains from the antiparallel extended form to the twisted one took place via an intermediate state. Many conformations revealed during the dynamics simulation showed that the relative orientations of each two Tyr1, Gly3, Phe4 and Met5 residues in the dimer are nearly related by a pseudo-C2-symmetry respectively, and both halves of the dimer structure could be further fitted to the monomeric folded enkephalin conformation. The monomeric and dimeric conformations of enkephalin at their stationary states are discussed in relation to the substrate-specificity for mu- and delta-opioid receptors.     

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.     

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.     

Survey of conformational role of ester bonds in a cyclic depsipeptide. Study on cyclo(-L-Ala-L-Hmb-)2 by energy calculation and NMR spectroscopy.

The effect of ester bond on the conformation of peptide molecule was studied by designing and synthesizing a model tetradepsipeptide cyclo(-L-Ala-L-Hmb-)2 and by analyzing the conformation both theoretically and experimentally. Theoretical analysis showed that both ester and peptide bonds in the calculated low-energy conformations within 3 kcal/mol of the global minimum take a trans but distorted configuration. The distortion is larger in ester bonds than in peptide bonds. Further, the four carbonyls project from one side of the plane of the cyclic backbone, whereas the side chains project from the other side. These results are consistent with the experimental results obtained by NMR measurement; first, the coupling constant deduced from 1H-NMR species in DMSO-d6 is consistent with the dihedral angles of the calculated low-energy conformations; second, results of NOE measurement can reproduce the calculated configuration of the carbonyls and side chains. From the consistency between theoretical and experimental results, it is concluded that this model tetradepsipeptide takes an all-trans backbone conformation in solution and this backbone conformation is stabilized by large distortion in the ester bond, which compensates the strain resulted from the 12-membered cyclic backbone structure consisting only of L-residues.     

Synthesis, biology, NMR and conformation studies of the topographically constrained delta-opioid selective peptide analogs of [beta-iPrPhe(3)]deltorphin I.

Replacement of Phe3 in the endogenous delta-opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid beta-isopropylphenylalanine (beta-iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand-binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [beta-iPrPhe3]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe3 residue in [beta-iPrPhe3]deltorphin I provided the most desirable biological properties with binding affinity (IC50 = 2 nM), bioassay potency (IC50 = 1.23 nM in MVD assay) and exceptional selectivity for the delta-opioid receptor over the mu-opioid receptor (30 000). Further conformational studies based on two-dimensional NMR and computer-assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr1 and (2S,3R)-beta-iPrPhe3 residues adopt trans side-chain conformations, and the linear peptide backbone favors a distorted beta-turn conformation.     

Hydration of peptides. I. Calculation of accessible surface areas for several conformations of a cyclic dipeptide

The concept of “static accessibility” to water has been used to determine the accessible surface area of a cyclic dipeptide: c(l-Thr-l-His). Different calculated and experimental conformations of this model molecule have been examined, which allows us to analyse the variations of accessibility of the hydration sites localized on the peptide backbone and on the polar side chains. The maximum solvation criterion involves a large destabilization of conformations governed by intramolecular interactions. The variations of the amphiphilic character with the conformations are relatively small. Nevertheless, the experimental conformation seems to reflect such a behaviour, especially in the crystal, in which the amphiphilic character is compatible with intermolecular interactions. The accessibility studies must be regarded only as a preliminary step to a more quantitative analysis of peptide hydration.     

Astressin-amide and astressin-acid are structurally different in dimethylsulfoxide

The C-terminally amidated CRF antagonist astressin binds to CRF-R1 or CRF-R2 receptors with low nanomolar affinity while the corresponding astressin-acid has >100 times less affinity. To understand the role of the amide group in binding, the conformations of astressin-amide and astressin-acid were studied in DMSO using NMR techniques. The 3D NMR structures show that the backbones of both analogs prefer an alpha-helical conformation, with a small kink around Gln(26). However, astressin-amide has a well-defined helical structure from Leu(27) to Ile(41) and a conformation very similar to the bioactive conformation reported by our group (Grace et al., Proc Natl Acad Sci USA 2007, 104, 4858-4863). In contrast, astressin-acid has an irregular helical conformation from Arg(35) onward, including a rearrangement of the side chains in that region. This structural difference highlights the crucial role of the C-terminal amidation for stabilization of astressin's bioactive conformation.     

Spatial structure of apamin in solution]

The calculation of the complete spatial structure of the bee venom peptide neurotoxin apamin has been carried out by means of a method elaborated earlier. It is based on the joint utilization of the molecular mechanics algorithms and NMR spectroscopy data. It was established that the molecule backbone conformation in solution may be represented as the combination of the beta-turn III (residues 2-5) and alpha-helical segment (9-18) both separated by the non-standard bend IV (5-8). The most probable system of the intramolecular hydrogen bonds in the apamin polypeptide backbone was proposed. Certain amino acid residues have been shown to be characterized by the lack of strict determination of the conformations of their side chains which may be realized in a few states providing approximately equal stabilization of the same form of the main chain. The conformational parameters of the proposed apamin structural model are appropriate to the NMR spectroscopy data derived from the literature and used in the calculations and are not contradictory to other experimental information.     

Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors. III. Probable binding conformations of mu-agonists with phenylalanine in position 3.

Theoretical conformational analysis was carried out for several tetrapeptide analogues of beta-casomorphin and dermorphin containing a Phe residue in position 3. Sets of low-energy backbone structures of the mu-selective peptides [N-Me-Phe3, D-Pro4]-morphiceptin and Tyr-D-Orn-Phe-Asp-NH2 were obtained. These sets of structures were compared for geometrical similarity between themselves and with the low-energy conformations found for the delta-selective peptide Tyr-D-Cys-Phe-D-Pen-OH and nonactive peptide Tyr-Orn-Phe-Asp-NH2. Two pairs of geometrically similar conformations of mu-selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the mu-receptor, were selected as two alternative models of probable mu-receptor-bound backbone conformations. Both models share geometrical similarity with the low-energy structures of the linear mu-selective peptide Tyr-D-Ala-Phe-Phe-NH2. Putative binding conformations of Tyr1 and Phe3 side chains are also discussed.     

Design, synthesis and conformational analysis of gamma-turn peptide mimetics of bradykinin.

Gamma-turns are regular secondary structure elements, found with some frequency in small peptides, that have been implicated in the biologically active conformations of several systems. This report describes the design, synthesis and conformational analysis of a non-peptide gamma-turn mimetic. Low energy conformations of the mimetic system exhibit good conformational agreement with an experimentally observed peptide gamma-turn. The mimetics were incorporated into the nonapeptide bradykinin, for which a gamma-turn, formed by residues Ser 6 to Phe 8, has been hypothesized to be a bioactive conformation. The results indicate that a bioactive conformation of bradykinin may include a reverse turn at this position.     

抗体片段在铂纳米粒子表面的构象重构

目的 最近在金纳米粒子（AuNPs）表面重构抗体片段的天然构象和功能的研究表明分子构象工程的可行性。本质上,分子构象工程就是要像蛋白质折叠一样,通过精确控制柔性非功能分子的构象使其产生新功能。本文在铂纳米粒子（PtNPs）表面重构抗体互补决定簇区（CDR）片段的天然构象和功能,旨在探索分子构象工程的普适性及揭示蛋白质结构-功能机制。方法 本文将抗溶菌酶抗体（cAB-lys3）中的CDR3片段（在单独存在时没有稳定构象和功能）通过两个Pt-S键偶联到PtNPs表面。CDR片段的天然构象和功能的恢复通过它对溶菌酶活性的抑制来表征。结果 通过多肽密度优化和表面聚乙二醇修饰,制得基于PtNPs的抗溶菌酶人工抗体（简称铂抗体）。溶菌酶活性测试结果表明,铂抗体可以特异性结合溶菌酶并显著抑制其活性。结论 本文第一次在PtNPs表面重构了蛋白质片段的天然构象并恢复了其功能,证明分子构象工程可作为一种通用方法制备基于纳米粒子的人工蛋白质。     

On the Bioactive Conformation of a Small Peptide and its Set of Thermodynamically Accessible Conformations

Abstract

In order to investigate the relationship between the bioactive conformation of a peptide and its set of thermodynamically accessible structures in solution, the conformational profile of the tetrapeptide Ac-Pro-Ala-Pro-Tyr-OH was characterized by computational methods. Search of the conformational space was performed within the molecular mechanics framework using the AMBER4.0 force field with an effective dielectric constant of 80. Unique structures of the peptide were compared with its bioactive conformation for the protein Streptomyces griseus Protease A, as taken from the crystal structure of the enzyme-peptide complex. The results show that the bound conformation is close to one of the unique conformations characterized in the conformational search of the isolated peptide. Moreover, the lowest energy minimum characterized in the conformational search exhibits large deviations when compared to the bound conformation of the crystal structure.     

Photomodulation of conformational states. II. Mono- and bicyclic peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to an ensemble of possible conformations.     

Stability of protein-bound conformations of bioactive peptides: the folded conformation of an epidermal growth factor-like thrombomodulin fragment is similar to that recognized by thrombin

The relationship between the free and bound conformations of bioactive peptides is explored using the epidermal growth factor (EGF)-like thrombomodulin fragment hTM409-426 as a model system. The hTM409-426 peptide has a sequence of C(409)PEGYILDDGFIC(421)TDIDE (with a disulfide bond between Cys409 and Cys421) and is a selective inhibitor of thrombin. Upon binding to thrombin, hTM409-426 adopts a well-defined conformation-namely, a beta-turn followed by an antiparallel beta-sheet, similar to those found in all other EGF-like protein repeats (Hrabal et al., Protein Science, 1996, Vol. 5, 195-203). Here we demonstrate that, at pH 6.8 and at 25 degrees C, the hTM409-426 peptide in the free state is very flexible, but still populates a type II beta-turn over residues Pro410-Glu411-Gly412-Tyr413 and the clustering of some hydrophobic side chains, both of which are present in the thrombin-bound conformation. At a lower temperature of 5 degrees C, significant conformational shifts of the C alpha H proton resonances and extensive medium- and long-range NOEs are observed, indicating the presence of folded conformations with unique backbone-backbone and side-chain interactions. A comparison of the NOE patterns in the free state with transferred NOEs shows that the free-state folded and the thrombin-bound conformations of the hTM409-426 peptide are very similar, particularly over residues Pro410-Ile424. The folded conformation of hTM409-426 appears to be stabilized by two hydrophobic clusters, one formed by the side chains of residues Pro410, Tyr413, Leu415, and Phe419 and the Cys409-Cys421 disulfide bond, the second involving residues Ile414 and Ile424. These results indicate that the overall topology of the thrombin-bound conformation of the hTM409-426 peptide is prefolded in the free state and the primary sequence (including the disulfide bond) may be selective for an ensemble of conformations similar to that recognized by thrombin.     

Preferred side‐chain conformation of arginine residues in a triple‐helical structure

The crystal structure of the triple‐helical peptide (Pro‐Hyp‐Gly)₃‐Pro‐Arg‐Gly‐(Pro‐Hyp‐Gly)₄ (POG3‐PRG‐POG4) was determined at 1.45 Å resolution. POG3‐PRG‐POG4 was designed to permit investigation of the side‐chain conformation of the Arg residues in a triple‐helical structure. Because of the alternative structure of one of three Arg residues, four side‐chain conformations were observed in an asymmetric unit. Among them, three adopt a ttg^?t conformation and the other adopts a tg^?g^?t conformation. A statistical analysis of 80 Arg residues in various triple‐helical peptides showed that, unlike those in globular proteins, they preferentially adopt a tt conformation for χ₁ and χ₂, as observed in POG3‐PRG‐POG4. This conformation permits van der Waals contacts between the side‐chain atoms of Arg and the main‐chain atoms of the adjacent strand in the same molecule. Unlike many other host–guest peptides, in which there is a significant difference between the helical twists in the guest and the host peptides, POG3‐PRG‐POG4 shows a marked difference between the helical twists in the N‐terminal peptide and those in the C‐terminal peptide, separated near the Arg residue. This suggested that the unique side‐chain conformation of the Arg residue affects not only the conformation of the guest peptide, but also the conformation of the peptide away from the Arg residue. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1000–1009, 2014.