title: Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex.
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.
authors.affiliation: Department of Cellular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MB-201, La Jolla, CA 92037, USA. abrooun@scripps.edu
authors.forename: Alexei
authors.initials: A
authors.lastname: Brooun
authors.forename: Scott A
authors.initials: SA
authors.lastname: Foster
authors.forename: Jill E
authors.initials: JE
authors.lastname: Chrencik
authors.forename: Ellen Y T
authors.initials: EY
authors.lastname: Chien
authors.forename: Anand R
authors.initials: AR
authors.lastname: Kolatkar
authors.forename: Markus
authors.initials: M
authors.lastname: Streiff
authors.forename: Paul
authors.initials: P
authors.lastname: Ramage
authors.forename: Hans
authors.initials: H
authors.lastname: Widmer
authors.forename: Gisbert
authors.initials: G
authors.lastname: Weckbecker
authors.forename: Peter
authors.initials: P
authors.lastname: Kuhn
mesh_terms: 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*
chemical_list: 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