ACTOBINDIN BINDS WITH HIGH-AFFINITY TO A COVALENTLY CROSS-LINKED ACTIN DIMER

Actobindin, a 9.8-kDa protein purified from Acanthamoeba castellanii, contains two actin-binding sites that can simultaneously bind two acti n monomers. However, actobindin inhibits actin polymerization to a gre ater extent than can be explained by its affinity for actin monomers ( site specific K-D = 3.3 mu M). This paradox would be resolved if actob indin could interfere with the nucleation phase of polymerization by u sing both binding sites to bind simultaneously to an actin oligomer be cause the interaction with oligomer would be thermodynamically favored over that with actin monomer. We now show that a covalently cross-lin ked actin dimer prepared from cross linked F-actin binds to actobindin with high affinity (apparent K-D = 11 nM) in accordance with theoreti cal predictions for simultaneous binding of two actin subunits per sin gle actobindin and consistent with the hypothesis that actobindin migh t bind to native actin oligomers and prevent them from nucleating poly merization. Furthermore, the interaction with cross-linked dimer exhib its specificity in that an isomeric cross-linked actin dimer with more rapid electrophoretic mobility binds weakly to actobindin. However, o nly this isomeric dimer is produced when cross-linking reagents are ad ded to actin undergoing polymerization in the presence of actobindin. Therefore, if actobindin inhibits polymerization by interacting with a native dimer whose conformation is similar to that of the cross-linke d dimer with slower electrophoretic mobility, then actobindin must eit her block the cross linking sites or convert the dimer to a different conformation.