title: The actin-binding domain of cortactin is dynamic and unstructured and affects lateral and longitudinal contacts in F-actin.
abstract: Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism, and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain [Zhang et al., 2007, Mol Cell 27:197-213]. We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0-9.6 A). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of intersubunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics.
authors.affiliation: Department of Chemistry and Biochemistry and Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA. alexs@ucla.edu
authors.forename: Alexander
authors.initials: A
authors.lastname: Shvetsov
authors.forename: Emir
authors.initials: E
authors.lastname: Berkane
authors.forename: David
authors.initials: D
authors.lastname: Chereau
authors.forename: Roberto
authors.initials: R
authors.lastname: Dominguez
authors.forename: Emil
authors.initials: E
authors.lastname: Reisler
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D051356:Cortactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D051356:Cortactin; C495869:Cttn protein, mouse; D050505:Mutant Proteins