Modeling of Multi-Domain Assemblies in α-Actinin-4 Reveals How External Cues Regulate its Binding to Actin

Abstract

α-Actinin-4 (ACTN4) is an actin-crosslinking protein that is essential to a number of processes in non-muscle cells, including motility and maintenance of cell shape. The binding of ACTN4 to actin filaments is dynamically regulated by a variety of external cues such as tyrosine phosphorylation, binding of calcium ions, binding of phosphoinositides, and calpain proteolysis, but the underlying molecular mechanisms are poorly understood. To address this, we developed an atomic model for the full ACTN4 homodimer, whose end regions contain a novel ternary complex between the C-terminal calmodulin-like domain of one monomer, and the N-terminal actin-binding domain (ABD) and an adjacent helical “neck” region of the opposite antiparallel monomer. This assembly is predicted to reduce actin binding by blocking the binding site on the CH1 subdomain of the ABD, which is validated by mutagenesis experiments designed specifically to disrupt the complex. using this atomic model, we are able to rationalize changes in actin binding due to external effectors as changes in the assembly of the ternary complex. Integrating these structural insights into a network model has allowed us to make detailed predictions that are consistent with a broad set of semi-quantitative experimental results. The similarity between ACTN4 and other actin-binding proteins suggests that many of our findings will apply for this whole class of proteins.