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 actin monomers. However, actobindin inhibits actin polymerization to a greater extent than can be explained by its affinity for actin monomers (site-specific KD = 3.3 microM). This paradox would be resolved if actobindin could interfere with the nucleation phase of polymerization by using both binding sites to bind simultaneously to an actin oligomer because the interaction with oligomer would be thermodynamically favored over that with actin monomer. We now show that a covalently cross-linked actin dimer prepared from cross-linked F-actin binds to actobindin with high affinity (apparent KD = 11 nM) in accordance with theoretical predictions for simultaneous binding of two actin subunits per single actobindin and consistent with the hypothesis that actobindin might bind to native actin oligomers and prevent them from nucleating polymerization. Furthermore, the interaction with cross-linked dimer exhibits specificity in that an isomeric cross-linked actin dimer with more rapid electrophoretic mobility binds weakly to actobindin. However, only this isomeric dimer is produced when cross-linking reagents are added 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-linked dimer with slower electrophoretic mobility, then actobindin must either block the cross-linking sites or convert the dimer to a different conformation.
Bubb
M R
MR
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Lewis
M S
MS
Korn
E D
ED
D000048:Acanthamoeba; D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002500:Centrifugation, Isopycnic; D003432:Cross-Linking Reagents; D006037:Glyoxal / Q000031:analogs & derivatives; D008840:Microfilament Proteins; D011108:Polymers; D011485:Protein Binding; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000378:metabolism*; D011721:Pyrenes
D000199:Actins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D011108:Polymers; D015800:Protozoan Proteins; D011721:Pyrenes; C050869:actobindin protein, Acanthamoeba; C066961:pyrene glyoxal; D006037:Glyoxal