Integrin α2I domain recognizes type I and type IV collagens by different mechanisms

The collagens are recognized by the alpha I domains of the collagen receptor integrins, A common structural feature in the collagen-binding alpha I domains is the presence of an extra helix, named helix alpha C. However, its participation in collagen binding has not been shown. Here, we have deleted the helix alpha C in the alpha(2)I domain and tested the function of the resultant recombinant protein (Delta alpha C alpha(2)I) by using a real-time biosensor, The Delta alpha C alpha(2)I domain had reduced affinity for type I collagen (430 +/- 90 nM) when compared with wild-type alpha(2)I domain (90 +/- 30 nM), indicating both the importance of helix alpha C in type I collagen binding and that the collagen binding surface in alpha(2)I domain is located near the metal ion-dependent adhesion site. Previous studies have suggested that the charged amino acid residues, surrounding the metal ion-dependent adhesion site but not interacting with Mg2+, may play an important role in the recognition of type I collagen, Direct evidence indicating the participation of these residues in collagen recognition has been missing. To test this idea, we produced a set of recombinant alpha(2)I domains with mutations, namely D219A, D219N, D219R, E256Q, D259N, D292N, and E299Q, Mutations in amino acids Asp(219), Asp(259), Asp(292), and Glu(299) resulted in weakened affinity for type I collagen. When alpha(2) D219N and D292N mutations were introduced separately into alpha(2)beta(1) integrin expressed on Chinese hamster ovary cells, no alterations in the cell spreading on type I collagen were detected. However, Chinese hamster ovary cells expressing double mutated alpha(2) D219N/D292N integrin showed remarkably slower spreading on type I collagen, while spreading on type TV collagen was not affected. The data indicate that alpha(2)I domain binds to type I collagen with a different mechanism than to type IV collagen.