Synthetic, Register-Specific, AAB Heterotrimers to Investigate Single Point Glycine Mutations in Osteogenesis Imperfecta

Abstract

Osteogenesis imperfecta (OI) is a disease caused primarily by mutations of glycine in the standard (Xaa-Yaa-Gly)n repeat of a type I collagen triple helix. Type I collagen is an AAB heterotrimer which means that, depending on whether the A or B chain is mutated, the glycine substitution will appear once or twice. In this work we use designed axial charged pairs to self-assemble an AAB triple helix with controlled composition and register. We then substitute a single glycine of the B chain with alanine, serine, valine, aspartate, or arginine and assess the impact on the structure and folding of this OI mimic by CD, NMR, and restraint-guided modeling. We find that alanine and serine substitutions are tolerated, resulting in localized disruptions to the triple helix structure, while bulkier amino acids result in alternatively folded structures. This work demonstrates the potential of axial charged pairs to control the structure of low stability triple helices and also helps to elucidate the structure and folding challenges associated with OI-type mutations in collagen.

How electrostatic networks modulate specificity and stability of collagen

“…Based on the interactions probed in abc, complex salt-bridge networks involving three charged groups showed anticooperativity, with the strongest effect on residues participating in an axial KD interaction. For proteins in general, it has been proposed that the extent and direction of cooperativity in multibody salt-bridge networks is dependent on the angle, θ, between the central charged group and two interacting partners (46) (Figs. 3A and 4A).…”

“…When θ < 90°, the central charged residue can interact tightly with both partners, resulting in positive cooperativity. When θ > 90°, the central charge must adopt different conformations to optimally interact with one of the other partners, resulting in anticooperativity (46). For abc, the average θ for lateral DK interaction was ∼85.4 ± 13.5°, on the cusp of anticooperativity.…”

Control of Collagen Triple Helix Stability by Phosphorylation

“…Peptides were prepared as previously described. 23 In brief, solid-phase peptide synthesis with an FMOC protecting strategy was used to obtain crude peptide. In all cases the glycine of the fourth triplet was isotopically labeled with 15 N. The peptide was purified by reverse-phase high-pressure liquid chromatography and then lyophilized to give the pure peptide as a powder.…”

“…25 Molecular modeling was performed as previously reported. 23 Through analysis of 1 H, 15 N-NOESY-HSQC and NOESY spectra, NOEs of protons closely associated with the H− 15 N were used to generate a combination of positive (0−4 Å distance) and negative constraints (6−10 Å distance) (SI Tables S3−10). These constraints were then used in the Rosetta Relax application 26 to generate models.…”

Site‐Specific Isotopic Labeling (SSIL): Access to High‐Resolution Structural and Dynamic Information in Low‐Complexity Proteins

“…In an elegant example of SSIL, several collagen‐derived peptides containing ten consecutive copies of the proline–hydroxyproline–glycine tripeptide (POG) 10 were synthesized by moving the position of a single [ 15 N]Gly within the chain, extremely simplifying resulting NMR spectra . Using this strategy, the dynamics and the effects of mutations and phosphorylation on the structure and stability of the collagen triple helix were investigated. However, the systematic application of SSIL using peptide synthesis strategies presents several limitations, such as the cost of the protected isotopically labeled amino acids, the length of the peptides amenable to peptide synthesis, and the presence of contaminants when this length increases.…”