Disease-associated mutant α-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity

alpha-Actinin-4 is a widely expressed protein that employs an actinbinding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca2+-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 poclocytes 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 a-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca2+ 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 a-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT a-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of a-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 a-actinin and possibly other calponin homology domain proteins under physiological conditions.