Structural and functional characterization of human Iba proteins
Authors
- J.O. Schulze
- C. Quedenau
- Y. Roske
- T. Adam
- H. Schueler
- J. Behlke
- A.P. Turnbull
- V. Sievert
- C. Scheich
- U. Mueller
- U. Heinemann
- K. Buessow
Journal
- FEBS Journal
Citation
- FEBS J 275 (18): 4627-4640
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 Ca(2+). 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 Ca(2+). Furthermore, no binding of Ca(2+) up to 0.1 mm was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca(2+) 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 extensions.