Structural and Functional Characterization of Human Iba Proteins

Abstract

Iba2 is a homolog of ionized calcium binding adapter molecule 1 Iba1 , a 17 kDa protein that binds and cross links filamentous actin F actin and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F actin cross linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF hand motifs lacking bound Ca2 . Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultracentrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca2 . Furthermore, no binding of Ca2 up to 0.1 amp; 8195;mm was detected by equilibrium dialysis. Correspondingly, Iba EF hand motifs lack residues essential for strong Ca2 coordination. Sedimentation experiments and microscopy detected pronounced, indistinguishable F actin binding and cross linking activity of Iba1 and Iba2 with induction of F actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co localized with F actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we studied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extension