Impact of vascular Ehlers-Danlos Syndrome-associated Gly substitutions on structure, function, and mechanics using bacterial collagen.

Vascular Ehlers-Danlos syndrome (vEDS) arises from mutations in collagen-III, a major structural component of the extracellular matrix (ECM) in vascularized tissues, including blood vessels. Fibrillar collagens form a triple-helix that is characterized by a canonical (Gly-X-Y)n sequence. The substitution of another amino acid for Gly within this conserved repeating sequence is associated with several hereditary connective tissue disorders, including vEDS. The clinical severity of vEDS depends on the identity of the substituted amino acid and its location. In this study, we engineered recombinant bacterial collagen-like proteins (CLPs) with previously reported Gly→X (X=Ser or Arg) vEDS substitutions within the integrin-binding site. Employing a combination of biophysical techniques, enzymatic digestion assays, integrin binding affinity assays, and computational modeling, we assessed the impact of Gly→X substitutions on structure, stability, function, and mechanical properties. While constructs with Ser or Arg substitutions maintained a triple-helix structure, Arg substitution significantly reduced global thermal stability, heightened susceptibility to trypsin digestion, and altered integrin α2-inserted (α2I) domain binding. Molecular dynamics (MD) simulations also demonstrated distinct effects of different Gly substitutions on the triple-helix structure - Arg substitutions induced notable bulging at the substitution site and disrupted interchain hydrogen bonds compared to Ser substitutions. Additionally, steered MD simulations revealed that Arg substitution led to a significant decrease in the Young's modulus of the triple-helix. Bacterial CLPs have proved to be a powerful model for studying the underlying mechanisms of vEDS-causing mutations in collagen-III. Serine and arginine substitutions differentially perturb cell-matrix interactions and ECM in a manner consistent with clinical vEDS severity.