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

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

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Actinin / chemistry*
  • Actinin / genetics
  • Actinin / metabolism
  • Actins / metabolism*
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Binding Sites
  • Calcium-Binding Proteins / metabolism
  • Humans
  • Microfilament Proteins / metabolism
  • Molecular Dynamics Simulation*
  • Molecular Sequence Data
  • Mutation, Missense
  • Phenotype
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Multimerization*

Substances

  • ACTN4 protein, human
  • Actins
  • Calcium-Binding Proteins
  • Microfilament Proteins
  • calponin
  • Actinin