Catch bonds, a class of noncovalent protein-protein interactions that paradoxically strengthen in the presence of mechanical force, are ubiquitous in cellular force transduction pathways mediating immunity, development, and tissue homeostasis. The structural mechanisms of catch bond formation remain unclear, hampering efforts to decipher how catch bonding proteins are dysregulated in disease and to exploit them therapeutically. The cytoskeletal crosslinker α-actinin-4 (ACTN4) forms catch bonds with actin filaments (F-actin), supporting the mechanical integrity of kidney podocytes as they are exposed to hemodynamic forces during blood filtration. The ACTN4 mutation K255E disrupts catch bonding activity by increasing the protein’s baseline F-actin binding affinity in the absence of force, causing autosomal dominant focal segmental glomerulosclerosis by rendering podocytes mechanically brittle. Leveraging a neural network-based approach for full-length cytoskeletal crosslinkers, we obtained cryo-EM structures of wild-type and K255E ACTN4 bound to F-actin. K255E ACTN4 engages F-actin in a single, stable conformation. In cryo-EM experiments on wild-type ACTN4, we see the emergence of a second conformation that is highly flexible. Biochemical experiments and molecular dynamics simulations support assigning these states as strongly bound and weakly bound, respectively. We next implemented a procedure for reconstituting physiological forces on cryo-EM grids using myosin motor proteins, uncovering a population shift from the weakly bound to the strongly bound state for wild-type ACTN4 in the presence of motor forces. These data are consistent with a previously proposed “two-state” model for catch bond formation, in which force promotes a weak-to-strong binding transition. In addition to providing insight into how the K255E disease mutation disrupts mechanical regulation of ACTN4, this study provides a framework for more broadly dissecting the mechanisms of cytoskeletal catch bonding proteins.
Chin et al. (Sun,) studied this question.