Structural and functional characterization of human Iba proteins.
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 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 extensions.
Schulze
Jörg O
JO
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Quedenau
Claudia
C
Roske
Yvette
Y
Adam
Thomas
T
Schüler
Herwig
H
Behlke
Joachim
J
Turnbull
Andrew P
AP
Sievert
Volker
V
Scheich
Christoph
C
Mueller
Uwe
U
Heinemann
Udo
U
Büssow
Konrad
K
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012760:Shigella / Q000472:pathogenicity
C112986:AIF1 protein, human; C532585:AIF1L protein, human; D000199:Actins; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D008840:Microfilament Proteins; D002118:Calcium