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 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.
Kreishman-Deitrick
Mara
M
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Goley
Erin D
ED
Burdine
Lyle
L
Denison
Carilee
C
Egile
Coumaran
C
Li
Rong
R
Murali
Nagarajan
N
Kodadek
Thomas J
TJ
Welch
Matthew D
MD
Rosen
Michael K
MK
D051376:Actin-Related Protein 2-3 Complex / Q000737:chemistry* / Q000528:radiation effects; D051378:Actin-Related Protein 3 / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010777:Photochemistry; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000737:chemistry* / Q000528:radiation effects
D051376:Actin-Related Protein 2-3 Complex; D051378:Actin-Related Protein 3; D003432:Cross-Linking Reagents; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal