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

Abstract

␣-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 Ca 2؉ -sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the ␣-actinin-4 actin-binding domain. Here we show that ␣-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 ␣-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 ␣-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT ␣-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of ␣-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 ␣-actinin and possibly other calponin homology domain proteins under physiological conditions. cytoskeleton ͉ kidney ͉ podocyte ͉ glomerulosclerosis

Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton

“…A mutation of lysine-255 to glutamate (K255E) in α-actinin 4 disrupts this salt bridge, driving the molecule into a permanently open configuration and revealing a third actin-binding site. In vitro, the K255E mutant has a five-fold higher actin binding affinity than the wild-type protein [30,31]. We hypothesized that network stress disrupts the salt bridge and converts the protein into the open, high affinity conformation, giving rise to catch-bond behavior of WT α-actinin 4 and leading to localized, stress-dependent accumulation.…”

Patients with ACTN4 Mutations Demonstrate Distinctive Features of Glomerular Injury

“…One possibility stems from recent evidence that mutant ␣-actinin-4 exhibits increased actinbinding affinity. 4,12 It is conceivable that altered ␣-actinin-4 binding affinity for actin could lead to a shift in equilibrium between bound and unbound actin, such that bound actin accumulates (perhaps slowly) in the podocyte cytoplasm. Over time, this material may accumulate to form the ultrastructurally visible aggregates, which ultimately contribute to podocyte damage or increased susceptibility to injury through interference with normal structure and function.…”

Filamin structure, function and mechanics: are altered filamin-mediated force responses associated with human disease?

“…α-actinin 4 missense mutations underlie the autosomal dominant kidney disorder, focal segmental glomerulosclerosis (Kaplan et al 2000), and are associated with increased F-actin affinity leading to cross-linking at a lower concentration of α-actinin 4 (Weins et al 2007). The viscoelastic properties of mutant α-actinin 4 cross-linked Factin networks show relaxation frequencies lowered by an order of magnitude owing to a reduced off rate of the mutant compared to wild-type (Ward et al 2008).…”