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 α2β1 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 → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 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 α2-inserted (α2I) 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 → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ 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.
Highlights
Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components
Several binding domains for critical ligands have been determined along the collagen III sequence [47], including those for von Willebrand factor [48], glycoprotein VI [49], integrins ␣11 and ␣21 [28, 29], kindlin-3 [50], discoidin domain receptor-2 [51], osteoclast-associated receptor [52], platelet fibrinogen receptor [53], leukocyte-associated immunoglobulin-like receptor-1 [54, 55], Yersinia adhesin A [56], heparin [57, 58], pigment epithelium– derived factor [59, 60], secreted protein acidic and rich in cysteines [61], decoron [62], and matrix metalloproteinases [63] (Figs. 1B and S1; integrin ␣21– binding motifs are highlighted in orange)
We observed that substitution of a Gly in the integrin-binding site interferes with ␣2I–collagen-mimetic peptides (CMPs) adhesion, dependent upon the identity of the Gly substitution, decreasing in the order WT Ͼ G240A Ͼ G240R Ͼ G240V, and T3-237 G240V substantially reduces human mesenchymal stem cells (hMSCs) spreading functionality relative to WT or G240A
Summary
The Ehlers–Danlos Syndrome Variant Database reports 611 cases of Gly 3 Xaa mutations that result in vEDS (https://eds. gene.le.ac.uk, updated May 14, 2019) [97, 98]. Each of the triple-helix ␣-chains of the mutated residues, Ala and Val, show considerable temperature-dependent chemical shift changes This indicates that even the small mutation of Gly 3 Ala abolishes the local hydrogen-bond network. We found that the triple helix is expanded, relative to the WT CMP, at each position that includes a Gly 3 Xaa mutation for all variants, and the effect asymmetrically propagates at least three positions C-terminal of the mutation sites, impacting functional domains. We have confirmed that the proper Mg2ϩ coordination and the van der Waals contacts, hydrogen bonds, and salt bridges that have been reported previously [29, 75] are present and stable in the simulated complex formed with the WT CMP (Table 1) These interactions remain unchanged upon introduction of an Ala mutation in the Xaa position,. This extreme loss in ␣2I–Mg2ϩ coordination in the case of the T3-237 G240V complex provides an explanation for the greatest reduction in ␣2I adhesion to T3-237 G240V
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