Common Interruptions in the Repeating Tripeptide Sequence of Non-fibrillar Collagens: Sequence Analysis and Structural Studies on Triple-helix Peptide Models

Abstract

Interruptions in the repeating (Gly-X1-X2) n amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher order structure and function. This report defines the sequence features and structural consequences of the most common interruption, where one residue is missing in the tripeptide pattern, Gly-X1-X2-Gly-AA 1 -Gly-X1-X2, designated as G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when a Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple-helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated as a G4G type, in that both are destabilizing, but allow continuation of rod-like triple-helices and maintenance of the 1-residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.

Triple‐helical peptides: An approach to collagen conformation, stability, and self‐association

“…Some interruptions are known to play a role in binding to tumor cell integrins 12 or as specific sites of matrix metalloproteinase cleavage, 33 and they have been suggested to represent flexible or kink sites involved in the network structure of Type IV collagen. [34][35][36] A total of 354 interruptions are found in all human nonfibrillar collagens and these can been classified according to number of residues within the interruption which are flanked by (Gly-X-Y) n triplets 37,38 (Figure 3). The repeating (Gly-X-Y) n sequence generates a pattern with two amino acids between Gly residues.…”

“…[37][38][39] Peptides can form stable triple-helices in presence of interruptions which have 1, 4, or 6 amino acids between Gly residues, but the break leads to decreased stability, decreased triple-helix content, and decreased hydrogen bonding. The identity of the residue in the middle and the nature of the surrounding Gly-X-Y sequence affects the degree of destabilization for the smallest interruptions.…”

“…Interruptions with one or four amino acids between Gly residues show similar effects on the thermal stability and calorimetric enthalpy, and NMR and X-ray structures indicate that both can be incorporated into a straight triple-helix with a highly localized distortion that disrupts the register of the helix. [38][39][40] However, NMR studies show that in a peptide with a GlyPro-Hyp-Gly-Phe-Gly-Pro-Hyp interruption, the hydrophobic residue Phe is located on the outside of the helix 38 ( Figure 4a), while a peptide with the interruption Gly-ProHyp-Gly-Ala-Ala-Val-Met-Gly-Pro-Hyp has the hydrophobic Val residue located on the inside of the triple-helix, forming a small hydrophobic core in the center of triple-helix usually occupied by Gly (Figure 4b). 39 The disruption of the axial register of the triple-helix by such interruptions may …”

“…38,39 In (a), the cross-section view shows the Gly residues are closely packed at the center with the Phe residues on the outside, whereas in (b) the Val packs in the center at the position usually occupied by Gly, creating a small hydrophobic core. (c) View of the distribution of the eight interruptions present in human Type X collagen.…”

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

“…The perturbations caused by 1-and 4-residue imperfections have been characterized in model peptides by X-ray crystallography and NMR spectroscopy. [15][16][17] These triple-helical peptides show distortion in dihedral angles and perturbed hydrogen bonding localized to the interruption site. In addition, the twist of the superhelix on one end of the peptide is out of register with the other end.…”