Formation and implications of a ternary complex of profilin, thymosin beta 4, and actin.
Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin beta(4), and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin beta(4) (residues 6-22) confirm the presence of an extensive binding surface between actin and thymosin beta(4), and explain why thymosin beta(4) and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin beta(4) peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin beta(4). Intracellular concentrations of thymosin beta(4) and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin beta(4)-actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin beta(4) can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.
Yarmola
E G
EG
Department of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Parikh
S
S
Bubb
M R
MR
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D003285:Contractile Proteins; D005454:Fluorescence Polarization; D008840:Microfilament Proteins / Q000378:metabolism*; D051304:Profilins; D011485:Protein Binding; D011817:Rabbits; D013947:Thymosin / Q000378:metabolism*; D014461:Ultracentrifugation; D000099201:Thymosin beta(4)
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D013947:Thymosin; D000099201:Thymosin beta(4)