title: Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity.
abstract: Alpha-actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca(2+)-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the alpha-actinin-4 actin-binding domain. Here we show that alpha-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant alpha-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca(2+) regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant alpha-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT alpha-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of alpha-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of alpha-actinin and possibly other calponin homology domain proteins under physiological conditions.
authors: Renal and Translational Medicine Divisions, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
authors: Astrid
authors: A
authors: Weins
authors: Johannes S
authors: JS
authors: Schlondorff
authors: Fumihiko
authors: F
authors: Nakamura
authors: Bradley M
authors: BM
authors: Denker
authors: John H
authors: JH
authors: Hartwig
authors: Thomas P
authors: TP
authors: Stossel
authors: Martin R
authors: MR
authors: Pollak
mesh_terms: D000185:Actinin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
chemical_list: C111453:ACTN4 protein, human; D000199:Actins; C491752:Actn4 protein, mouse; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D000185:Actinin