Structure of collagen receptor integrin α 1I domain carrying the activating mutation E317A

Abstract

We have analyzed the structure and function of the integrin α 1I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α 1I C139S/E317A was a higher avidity collagen binder than α 1I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α 1I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α 2I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α 2I structure, has not changed its position in the activated α 1I variant. During the integrin activation, Glu 335 on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β 1 subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu 335. This indicates that the stabilization of helix 7 into its downward position is not required if the α 1 MIDAS is already open. To conclude, the activated α1 I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg 287-Glu 317 ion pair has just broken during the integrin activation. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.