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.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Proteomics , Proto-Oncogene Proteins c-vav/isolation & purification , Proto-Oncogene Proteins c-vav/metabolism , rac1 GTP-Binding Protein/metabolism , Amino Acid Sequence , Cloning, Molecular , Crystallization , Cysteine/chemistry , DNA, Complementary , Glutathione Transferase/metabolism , Guanine Nucleotide Exchange Factors/analysis , Hydrolysis , Kinetics , Molecular Sequence Data , Mutagenesis , Nanotechnology , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Proteins/metabolism , Proto-Oncogene Proteins c-vav/chemistry , Proto-Oncogene Proteins c-vav/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Zinc/chemistryABSTRACT
Rhythmic behaviors are a fundamental feature of all organisms. Pharyngeal pumping, the defecation cycle, and gonadal-sheath-cell contractions are three well-characterized rhythmic behaviors in the nematode C. elegans. The periodicities of the rhythms range from subsecond (pharynx) to seconds (gonadal sheath) to minutes (defecation). However, the molecular mechanisms underlying these rhythmic behaviors are not well understood. Here, we show that the C. elegans Rho/Rac-family guanine nucleotide exchange factor, VAV-1, which is homologous to the mammalian Vav proto-oncogene, has a crucial role in all three behaviors. vav-1 mutants die as larvae because VAV-1 function is required in the pharynx for synchronous contraction of the musculature. In addition, ovulation and the defecation cycle are abnormal and arrhythmic. We show that Rho/Rac-family GTPases and the signaling molecule inositol triphosphate (IP(3)) act downstream of VAV-1 signaling and that the VAV-1 pathway modulates rhythmic behaviors by dynamically regulating the concentration of intracellular Ca(2+).
