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

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 label...

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Published in:Biochemistry (Easton) 2005-11, Vol.44 (46), p.15247-15256
Main Authors: Kreishman-Deitrick, Mara, Goley, Erin D, Burdine, Lyle, Denison, Carilee, Egile, Coumaran, Li, Rong, Murali, Nagarajan, Kodadek, Thomas J, Welch, Matthew D, Rosen, Michael K
Format: Article
Language:English
Subjects:
Actin-Related Protein 2-3 Complex - chemistry
Actin-Related Protein 2-3 Complex - radiation effects
Actin-Related Protein 3 - chemistry
Amino Acid Sequence
Animals
Cattle
Cross-Linking Reagents - chemistry
Humans
Molecular Sequence Data
Nuclear Magnetic Resonance, Biomolecular
Photochemistry
Wiskott-Aldrich Syndrome Protein, Neuronal - chemistry
Wiskott-Aldrich Syndrome Protein, Neuronal - radiation effects
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi051065n