Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Abstract

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin ␣ 2 ␤ 1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly 3 Xaa (Xaa ‫؍‬ Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin ␣ 2 ␤ 1binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin ␣ 2 ␤ 1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin ␣ 2-inserted (␣ 2 I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly 3 Xaa substitutions differentially disrupt the CMP-␣ 2 I interaction. The Gly 3 Arg substitution destabilized CMP-␣ 2 I side-chain interactions, and the Gly 3 Val change broke the essential Mg 2؉ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Purification of recombinant bacterial collagens containing structural perturbations

“…However, the introduction of Gly→Arg within the IBS of bacterial collagen, VCL 2 (G→R)-Trypsin, increased sensitivity to trypsin digestion with detectable CL 2 cleavage products after only 2 minutes ( Fig 5B ). This is consistent with the highly disruptive nature of Gly→Arg substitutions within the triple helix [ 22 , 28 – 30 ]. After overnight digestion with trypsin, the VCL 2 (G→R)-Trypsin protein is entirely degraded, hampering purification and significantly reducing the final yield of CL 2 (G→R).…”

Production and Functional Analysis of Collagen Hexapeptide Repeat Sequences in Pichia pastoris

“…To investigate the specific effect of metal ions on the binding of the structural domain of α 2 I, we performed ELISA experiments . α 2 I interaction with collagen depends on the coordination of divalent metal cations to Glu in the recognition structural domain. , Therefore, binding assays were performed in the presence of Mg 2+ and Ca 2+ and in the absence of both cations to control for nonspecific binding.…”

Designing collagens to shed light on the multi-scale structure–function mapping of matrix disorders

“…For example, CMPs were designed to include reported Gly → X (X = Ala, Arg, and Val) substitutions occurring within the high-affinity integrin α2β1 binding motif, GROGER, in vascular EDS patients. These mutated sites were flanked with (GPO) n sequences at both ends [138] . The study again demonstrated that the identity of the residue in a Gly → X substitution could have a range of impacts on triple helix stability, binding with the integrin α2-inserted (α2-I) domain, and the expected severity of a disease phenotype [138] .…”

“…These mutated sites were flanked with (GPO) n sequences at both ends [138] . The study again demonstrated that the identity of the residue in a Gly → X substitution could have a range of impacts on triple helix stability, binding with the integrin α2-inserted (α2-I) domain, and the expected severity of a disease phenotype [138] . Cell adhesion assays demonstrated that human mesenchymal stem cells (hMSCs) grown on the Gly → Ala peptide exhibited strong cell adhesion similar to wild-type peptides, while cells cultured on Gly → Val peptide adhered poorly, and those that were adherent displayed altered morphology with a “pancake-like” appearance [138] .…”