Web Release Date: October 29,
NMR Analyses of the Activation of the Arp2/3 Complex by Neuronal
Wiskott-Aldrich Syndrome Protein
and
Department of Biochemistry and Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, Texas 75390, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, and Varian Instruments, Palo Alto, California 94304
Received June 4, 2005
Revised Manuscript Received September 28, 2005
Abstract:
The VCA domain of the neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a potent activator of the Arp2/3 complex, a 240 kDa heteroheptameric actin-nucleating assembly. We used site-directed spin labeling of N-WASP peptides in conjunction with methyl-TROSY spectra of the intact, selectively labeled Arp2/3 complex to identify regions of the VCA that are proximal to the ARPC3 subunit of the assembly. We also cross-linked CA peptides to the Arp3, Arp2, ARPC1, and ARPC3 subunits. The combined data suggest that the extreme C-terminus of the A region and the C-terminus of the C region of N-WASP are proximal to ARPC3. These results have implications for the mechanism of Arp2/3 complex activation by VCA peptides. This study also demonstrates the utility of NMR spectroscopy for studying ligand binding events in large, asymmetric, macromolecular assemblies.
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