Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting.
Cofilin is an actin regulatory protein that binds to both monomeric and filamentous actin, and has filament severing activity. Although crystal structures for the monomeric forms of both G-actin and cofilin have been described, the structure of the binary cofilin-G-actin complex is not available. Synchrotron protein footprinting is used to identify specific side chain residues on the cofilin surface that are buried in the formation of the cofilin-G-actin binary complex. Exposure to synchrotron X-rays results in stable oxidative modifications of aromatic, aliphatic, and sulfur-containing side chains, with the rate of modification for a particular residue being dependent on its intrinsic reactivity and solvent accessibility. The rates of modification were monitored for a number of peptides generated by digestion of oxidized cofilin, both in isolation and in its binary complex with G-actin. After binding to G-actin takes place, a significant decrease in modification rates, indicating protection of side chain groups, is seen for cofilin peptides corresponding to residues 4-20, 10-17, 83-96, 91-105, and 106-117. A number of other peptides show no change in reactivity, and are presumed to represent regions distal to the binding site. Tandem mass spectrometry demonstrates that residues Leu 13, Pro 94, Met 99, and Leu 108 and 112 directly participate in the binding interface. These results are generally consistent with, and complementary to, the results of previous site-directed mutagenesis studies and extend our understanding of the G-actin binding surface of cofilin.
Guan
Jing-Qu
JQ
Center for Synchrotron Biosciences, Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Vorobiev
Sergeui
S
Almo
Steven C
SC
Chance
Mark R
MR
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D023302:Protein Footprinting; D011817:Rabbits; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017356:Synchrotrons; D014965:X-Rays; D015003:Yeasts
D051339:Actin Depolymerizing Factors; D000199:Actins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D010446:Peptide Fragments; D012997:Solvents