Journal
BIOPHYSICAL JOURNAL
Volume 104, Issue 3, Pages 705-715Publisher
CELL PRESS
DOI: 10.1016/j.bpj.2012.12.003
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Funding
- NlH [R01 GM97082, R01 GM69668]
- T32 Training Grant [5T32EB009403-02]
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The assembly of proteins into multidomain complexes is critical for their function. In eukaryotic nonmuscle cells, regulation of the homodimeric actin cross-linking protein alpha-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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