Article

Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex.

Department of Cellular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MB-201, La Jolla, CA 92037, USA.
Protein Expression and Purification (impact factor: 1.59). 06/2007; 53(1):51-62. DOI:10.1016/j.pep.2006.10.027 pp.51-62
Source: PubMed

ABSTRACT The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here, we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.

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Keywords

1D NMR analysis
 
cysteine-rich domain
 
GEF/GTPase pairs
 
ICP-MS analysis
 
isolated CRD
 
key roles
 
lymphocyte activation
 
remedial structural proteomics strategies
 
signal transduction pathway
 
stable Vav1
 
structural characterization
 
three dimensional Vav1/Rac1 complex crystal structure
 
unique protein-protein interface
 
various Vav1 domains
 
Vav1 guanine nucleotide exchange factor
 
Vav1-DH-PH-CRD
 
Vav1-DH-PH-CRD/Rac1 complex
 
Vav1/Rac1 complex suitable
 
Vav1/Rac1 protein-protein interaction
 
vitro binding experiments