Integrin alpha I-2 domain recognizes type I and type IV collagens by different mechanisms

The collagens are recognized by the alpha I domains of the collagen recepto r integrins, A common structural feature in the collagen-binding alpha I do mains 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 r esultant 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 typ e 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 c ollagen binding and that the collagen binding surface in alpha(2)I domain i s 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 importan t role in the recognition of type I collagen, Direct evidence indicating th e 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, Mut ations in amino acids Asp(219), Asp(259), Asp(292), and Glu(299) resulted i n weakened affinity for type I collagen. When alpha(2) D219N and D292N muta tions 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 d ouble mutated alpha(2) D219N/D292N integrin showed remarkably slower spread ing on type I collagen, while spreading on type TV collagen was not affecte d. The data indicate that alpha(2)I domain binds to type I collagen with a different mechanism than to type IV collagen.