Covalent binding of ATPgammaS to the nucleotide-binding site in S14C-actin.
We have recently reported on the characterization of beta-actin carrying the mutation S14C in one of the phosphate-binding loops. The present paper describes the attachment of the adenosine 5'-[gamma-thio]-triphosphate (ATPgammaS) to actin containing this mutation. Treatment of S14C-actin with ATPgammaS blocked further nucleotide exchange and raised the thermal stability of the protein, suggesting the formation of a covalent bond between the sulfhydryl on the terminal phosphate of ATPgammaS and cysteine-14 of the mutant actin. The affinity of the derivatized G-actin for DNase I as compared to wild-type ATP-actin was lowered to a similar extent as that of ADP.AlF(4)-actin. The derivatized actin polymerized slower than ATP-actin but faster than ADP-actin. Under these conditions the bound ATPgammaS was hydrolyzed, suggesting the formation of a state corresponding to the transient ADP.P(i)-state. ATPgammaS-actin interacted normally with profilin, whereas the interaction with actin depolymerizing factor (ADF) was disturbed, as judged on the effects of these proteins on actin polymerization.
Schüler
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Department of Cell Biology, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
Schutt
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CE
Lindberg
U
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Karlsson
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D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D002645:Chickens; D003285:Contractile Proteins; D004355:Drug Stability; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051304:Profilins; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000199:Actins; D001704:Biopolymers; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate