|Guan, J. Q., Chance, M. R.
& Almo, S. C. (2001) Mapping the binding sites of yeast cofilin and
G-actin by X-ray protein footprinting. Biophysical Journal. 80,
|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.