Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex.
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.
Brooun
Alexei
A
Department of Cellular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MB-201, La Jolla, CA 92037, USA. abrooun@scripps.edu
Foster
Scott A
SA
Chrencik
Jill E
JE
Chien
Ellen Y T
EY
Kolatkar
Anand R
AR
Streiff
Markus
M
Ramage
Paul
P
Widmer
Hans
H
Weckbecker
Gisbert
G
Kuhn
Peter
P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D036103:Nanotechnology; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D040901:Proteomics; D051567:Proto-Oncogene Proteins c-vav / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000737:chemistry; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D051567:Proto-Oncogene Proteins c-vav; D011994:Recombinant Proteins; C496618:VAV1 protein, human; D005982:Glutathione Transferase; D020830:rac1 GTP-Binding Protein; D015032:Zinc; D003545:Cysteine