Kenji Okuyama

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.

The single‐crystal structure of the collagen‐like peptide (Pro‐Pro‐Gly)₄‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)₄, was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple‐helical structure and that its conformation is very similar to that of (Gly‐Pro‐Hyp)₉, which has the typical repeating sequence in collagen. High‐resolution studies on other collagen‐like peptides have shown that imino acid‐rich sequences preferentially adopt a 7/2 triple‐helical structure (θ = 51.4°), whereas imino acid‐lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly‐Hyp‐Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro‐Pro‐Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2‐helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N‐terminus of an adjacent molecule, causing axial displacement, reminiscent of the D‐staggered structure in fibrous collagens. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 436–447, 2013.

Collagens have long been believed to adopt a triple‐stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X‐ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single‐crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly‐Pro‐Hyp)₉ peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly‐Pro‐Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly‐Pro‐Hyp)₉ peptide adopts a triple‐stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 607–616, 2012.

The single‐crystal structures of three collagen‐like host–guest peptides, (Pro‐Pro‐Gly)₄‐Hyp‐Yaa‐Gly‐(Pro‐Pro‐Gly)₄ [Yaa = Thr, Val, Ser; Hyp = (4R)‐4‐hydroxyproline] were analyzed at atomic resolution. These peptides adopted a 7/2‐helical structure similar to that of the (Pro‐Pro‐Gly)₉ peptide. The stability of these triple helices showed a similar tendency to that observed in Ac‐(Gly‐Hyp‐Yaa)₁₀‐NH₂ (Yaa = Thr, Val, Ser) peptides. On the basis of their detailed structures, the differences in the triple‐helical stabilities of the peptides containing a Hyp‐Thr‐Gly, Hyp‐Val‐Gly, or Hyp‐Ser‐Gly sequence were explained in terms of van der Waals interactions and dipole–dipole interaction between the Hyp residue in the X position and the Yaa residue in the Y position involved in the Hyp(X):Yaa(Y) stacking pair. This idea also explains the inability of Ac‐(Gly‐Hyp‐alloThr)₁₀‐NH₂ and Ac‐(Gly‐Hyp‐Ala)₁₀‐NH₂ peptides to form triple helices. In the Hyp(X):Thr(Y), Hyp(X):Val(Y), and Hyp(X):Ser(Y) stacking pairs, the proline ring of the Hyp residues adopts an up‐puckering conformation, in agreement with the residual preference of Hyp, but in disagreement with the positional preference of X in the Gly‐Xaa‐Yaa sequence. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 628–640, 2011.

Two crystal modifications of a collagen model peptide, (Pro-Pro-Gly)₄-Hyp-Hyp-Gly-(Pro-Pro-Gly)₄[where Hyp is (4R,2S)-L-hydroxyproline], showed very similar unit-cell parameters and belonged to the same space groupP2₁. Both crystals exhibited pseudo-merohedral twinning. The main difference was in their molecular-packing arrangements. One modification showed pseudo-hexagonal packing, while the other showed pseudo-tetragonal packing. Despite their different packing arrangements, no significant differences were observed in the hydration states of these modifications. The peptide in the pseudo-tetragonal crystal showed a cyclic fluctuation of helical twists with a period of 20 Å, while that in the pseudo-hexagonal crystal did not. In these modifications, the puckering conformations of four of the 12 Hyp residues at theXposition of the Hyp(X)-Hyp(Y)-Gly sequence were in the opposite conformations to the previous hypothesis that Hyp(X) residues involved in Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs prefer up-puckering and down-puckering conformations, respectively. Detailed investigation of the molecular interactions between Hyp(X) and adjacent molecules revealed that these opposite conformations appeared because the puckering conformation, which follows the hypothesis, is subject to steric hindrance from the adjacent molecule.

Structures of (Pro‐Pro‐Gly)₄‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)₄ (ppg9‐XYG) where (Xaa, Yaa) = (Pro, Hyp), (Hyp, Pro) or (Hyp, Hyp) were analyzed at high resolution using synchrotron radiation. Molecular and crystal structures of these peptides are very similar to those of the (Pro‐Pro‐Gly)₉ peptide. The results obtained in this study, together with those obtained from related compounds, indicated the puckering propensity of the Hyp in the X position: (1) Hyp(X) residues involved in the Hyp(X):Pro(Y) stacking pairs prefer the down‐puckering conformation, as in ppg9‐OPG, and ppg9‐OOG; (2) Hyp(X) residues involved in the Hyp(X):Hyp(Y) stacking pairs prefer the up‐puckering conformation if there is no specific reason to adopt the down‐puckering conformation. Water molecules in these peptide crystals are classified into two groups, the 1st and 2nd hydration waters. Water molecules in the 1st hydration group have direct hydrogen bonds with peptide oxygen atoms, whereas those in the 2nd hydration group do not. Compared with globular proteins, the number of water molecules in the 2nd hydration shell of the ppg9‐XYG peptides is very large, likely due to the unique rod‐like molecular structure of collagen model peptides. In the collagen helix, the amino acid residues in the X and Y positions must protrude outside of the triple helix, which forces even the hydrophobic side chains, such as Pro, to be exposed to the surrounding water molecules. Therefore, most of the waters in the 2nd hydration shell are covering hydrophobic Pro side chains by forming clathrate structures. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 361–372, 2009.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The crystal structure of a collagen-model peptide [(Pro-Pro-Gly)₉]₃ has been determined at 1.33 Å resolution. Diffraction data were collected at 100 K using synchrotron radiation, which led to the first structural study of [(Pro-Pro-Gly)_n]₃ under cryogenic conditions. The crystals belong to the P2₁ space group with cell parameters of a = 25.95, b = 26.56, c = 80.14 Å and β = 90.0°. The overall molecular conformation was consistent with the left-handed 7/2-helical model with an axial repeat of 20 Å for native collagen. A total of 332 water molecules were found in an asymmetric unit. Proline residues in adjacent triple-helices exhibited three types of hydrophobic interactions. Furthermore, three types of hydrogen-bonding networks mediated by water molecules were observed between adjacent triple-helices. These hydrophobic interactions and hydrogen-bonding networks occurred at intervals of 20 Å along the c-axis based on the previous sub-cell structures [(Pro-Pro-Gly)_n]₃ (n = 9, 10), which were also seen in the full-cell structure of [(Pro-Pro-Gly)₁₀]₃. Five proline residues at the Y position in the X-Y-Gly triplet were found in a down-puckering conformation, this being inconsistent with the recently proposed propensity-based hypothesis. These proline residues were forced to adopt opposing puckering because of the prevailing hydrophobic interaction between triple-helices compared with the Pro:Pro stacking interaction within a triple-helix.