Cofilin induced conformational changes in F-actin expose subdomain 2 to proteolysis.
Cofilin/ADF affects strongly the structure of actin filaments and especially the intermolecular contacts of the DNase I binding loop (D-loop) in subdomain 2. In G-actin, the D-loop is cleaved by subtilisin between Met47 and Gly48, while in F-actin this cleavage is inhibited. Here, we report that yeast cofilin, which is resistant to both subtilisin and trypsin, accelerates greatly the rate of subtilisin cleavage of this loop in F-actin at pH 6.8 and at pH 8.0. Similarly, cofilin accelerates strongly the tryptic cleavage in F-actin of loop 60-69 in subdomain 2, at Arg62 and Lys68. The acceleration of the loops' proteolysis cannot be attributed to an increased treadmilling of F-actin for the following reasons: (i) the rate of subtilisin cleavage is independent of pH between pH 6.8 and 8.0, unlike F-actin depolymerization, which is pH-dependent; (ii) at high concentrations of protease the cleavage rate of F-actin in the presence of cofilin is faster than the rate of monomer dissociation from the pointed end of TRC-labeled F-actin, which limits the rate of treadmilling; and (iii) cofilin also accelerates the rate of subtilisin cleavage of F-actin in which the treadmilling is blocked by interprotomer cross-linking of the D-loop to the C terminus on an adjacent protomer. This suggests a substantial flexibility of the D-loop in the cross-linked F-actin. The increased cleavage rates of the D-loop and loop 60-69 reveal extensive exposure of subdomain 2 in F-actin to proteolytic enzymes by cofilin.
Muhlrad
Andras
A
Institute of Dental Sciences, School of Dental Medicine, Hebrew University of Jerusalem, P.O. Box 12272, Jerusalem 91120, Israel. muhlrad@cc.huji.ac.il
Kudryashov
Dmitry
D
Michael Peyser
Y
Y
Bobkov
Andrey A
AA
Almo
Steve C
SC
Reisler
Emil
E
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D020860:Subtilisin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D000199:Actins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D010447:Peptide Hydrolases; D014357:Trypsin; D020860:Subtilisin