Conformational Features of a Natural Break in the Type IV Collagen Gly-X-Y Repeat

Abstract

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the ␣5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with T m ‫؍‬ 29°C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10°C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15 N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly) 10 , (GPOG-POPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.The repeating (Gly-X-Y) n sequence of the collagen triple helix allows for easy identification of this structural motif from amino acid sequences. The collagen triple helix structure consists of three supercoiled polypeptide chains, each in a polyproline II-like conformation (1-4). The repeating pattern of Gly as every 3rd residue is generated by steric requirements of the close packing of the three supercoiled polypeptide chains near a central axis. The collagen amino acid sequence is also characterized by a high frequency of proline and hydroxyproline (Hyp 4 ) in the X and Y positions, respectively, which serves to stabilize the polyproline II-like helix of the individual chains. The collagen triple helix is the defining motif of all extracellular matrix proteins classified as collagens and is also found as a domain in various other proteins, including C1q, macrophage scavenger receptor, collagenous tail of asymmetric acetylcholinesterase, and the bacterial proteins Streptococcus pyogenes scl and Bacillus anthraces bcl1 (5-9).Although the (Gly-X-Y) n repeating sequence is a requirement to form a collagen triple helix, this pattern is perfectly maintained in some collagen domains but not in others (9). The most abundant collagens are those found in fibrils with a 670 Å axial period (types I, II, III, V, XI), and these all maintain a precise Gly-X-Y repeat throughout their ϳ1000-residue triple helix domain. The replacement of even one Gly by anot...

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.…”

Solution Structure of an ABC Collagen Heterotrimer Reveals a Single-register Helix Stabilized by Electrostatic Interactions

“…Monomeric samples (K and D) were analyzed exclusively through two-dimensional total correlated spectroscopy (TOCSY) 2 To determine the register of the triple helix, a two-dimensional version of a four-dimensional 1 H, 13 C-HMQC-NOESY-1 H, 15 N-HSQC experiment was recorded at 25°C (26); to ease further discussion of this experiment, it will be referred to as a two-dimensional 13 C, 15 N-edited NOESY. Details on this experiment are available in the supplemental material.…”

“…Such peptides have been used to study the structure (6, 7), folding (8), and dynamics (9) of the triple helix. These peptides have been shown to retain the biochemical properties of the higher assemblies found in their natural counterparts, binding to cell surface proteins such as integrins (10).Most of the studies performed on collagen mimetic peptides utilize triple helices with three identical chains called homotrimers (8,(11)(12)(13). Such systems are good models for some types of collagen, like type II.…”

“…Most of the studies performed on collagen mimetic peptides utilize triple helices with three identical chains called homotrimers (8,(11)(12)(13). Such systems are good models for some types of collagen, like type II.…”