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Biophys J. 2013 Feb 5;104(3):705-15. doi: 10.1016/j.bpj.2012.12.003.

Modeling the assembly of the multiple domains of α-actinin-4 and its role in actin cross-linking.

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Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.


The assembly of proteins into multidomain complexes is critical for their function. In eukaryotic nonmuscle cells, regulation of the homodimeric actin cross-linking protein α-actinin-4 (ACTN4) during cell migration involves signaling receptors with intrinsic tyrosine kinase activity, yet the underlying molecular mechanisms are poorly understood. As a first step to address the latter, we validate here an atomic model for the ACTN4 end region, which corresponds to a ternary complex between the N-terminal actin-binding domain (ABD) and an adjacent helical neck region of one monomer, and the C-terminal calmodulin-like domain of the opposite antiparallel monomer. Mutagenesis experiments designed to disrupt this ternary complex confirm that its formation reduces binding to F-actin. Molecular dynamics simulations show that the phosphomimic mutation Y265E increases actin binding by breaking several interactions that tether the two calponin homology domains into a closed ABD conformation. Simulations also show a disorder-to-order transition in the double phosphomimic mutant Y4E/Y31E of the 45-residue ACTN4 N-terminal region, which can inhibit actin binding by latching both calponin homology domains more tightly. Collectively, these studies provide a starting point for understanding the role of external cues in regulating ACTN4, with different phenotypes resulting from changes in the multidomain assembly of the protein.

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