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Article

NMR Analyses of the Activation of the Arp2/3 Complex by Neuronal Wiskott−Aldrich Syndrome Protein^†

Supporting Info

Mara Kreishman-Deitrick,^‡^§ Erin D. Goley, Lyle Burdine, Carilee Denison, Coumaran Egile,^# Rong Li,^# Nagarajan Murali, Thomas J. Kodadek, Matthew D. Welch, and Michael K. Rosen*^‡

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

Biochemistry, 2005, 44 (46), pp 15247–15256

DOI: 10.1021/bi051065n

Publication Date (Web): October 29, 2005

†

Supported by grants from the NIH (P01GM066311 to M.K.R. and R.L. and GM59609 to M.D.W.) and the Welch Foundation (I1544 to M. K. R.). E.D.G. is supported by an NSF graduate research fellowship.

‡

Department of Biochemistry, University of Texas Southwestern Medical Center.

Current address: Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910.

University of California, Berkeley.

Center for Biomedical Inventions, University of Texas Southwestern Medical Center.

Stowers Institute for Medical Research.

Varian Instruments.

To whom correspondence should be addressed. E-mail: mrosen@biochem.swmed.edu. Phone: 214-645-6361. Fax: 214-645-6353.

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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