title: Widely distributed residues in thymosin beta4 are critical for actin binding.
abstract: We have investigated the contributions of hydrophobic residues, the conserved and variable proline residues, and the conserved lysine residues to the affinity and kinetics of thymosin beta4 (Tbeta4) binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted with alanine residues. Mutagenesis of Pro4 or Pro27 has little effect (<or=3-fold reduction) on the actin binding affinity of Tbeta4. Substitution of Lys18 and Lys19, Leu28, Pro29, or Ile34 weakens the affinity of the actin-Tbeta4 complex >or=10-fold, but the kinetic basis of the lower stability varies among the mutants. Substitution of the conserved lysine residues weakens the affinity by slowing association and accelerating dissociation. Substitution of hydrophobic residue Leu28 or Ile34 weakens the affinity by accelerating dissociation. These results favor a reaction mechanism in which Tbeta4 binds actin monomers following a two-step mechanism in which the formation of a bimolecular complex is followed by isomerization to a strong binding state that is coupled to the formation of widely distributed hydrophobic contacts. The isomerization equilibrium is slowed by mutagenesis of Pro29, as revealed by the double-exponential time course of association. Mutagenesis of Pro4 or Pro27 accelerates binding and dissociation but minimally affects the binding affinity (<or=3-fold reduction), suggesting that cis- trans isomerization of these proline residues contributes to the slow association rate constant of wild-type Tbeta4.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, Connecticut 06520, USA.
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Au
authors.forename: Adrian O
authors.initials: AO
authors.lastname: Olivares
authors.forename: Arnon
authors.initials: A
authors.lastname: Henn
authors.forename: Wenxiang
authors.initials: W
authors.lastname: Cao
authors.forename: Daniel
authors.initials: D
authors.lastname: Safer
authors.forename: Enrique M
authors.initials: EM
authors.lastname: De La Cruz
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D008958:Models, Molecular; D011487:Protein Conformation; D011817:Rabbits; D013947:Thymosin / Q000737:chemistry / Q000378:metabolism*; D000099201:Thymosin beta(4)
chemical_list: D000199:Actins; D013947:Thymosin; D000099201:Thymosin beta(4)