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.

Figure 1

A) Vav1 domain boundaries. Vav1 consists of several domains, including a calmodulin homology domain (CH, 1-116), an acidic region (Ac, 132-176), a Dbl homology domain (DH; 185-375), a pleckstrin homology domain (PH, 398-508), a unique cysteine rich domain (CRD, 516-565), a short proline rich region (607-610), and an SH2-SH3-SH2 domain (612-844). B) Key elements of Vav1-DH-PH-CRD unit as related to hVav1 construct used for crystallization of Vav1-Rac1 complex. Y174 is regulatory tyrosine residue that controls opening/closure of DH domain. V8 protease cleaves after residue E175. Amino acids 170, 180 and 190 are starting points for the majority of Vav1 constructs in this study. Constructs starting from residue 170 are considered to be in the “closed conformation”, while constructs starting from residues 180 or 190 are considered to be in the “open conformation” and refers to the presence or absence of a N-terminal autoinhibitory helix.

Figure 2

Microexpression analysis of hVav DH to mVav DH mutants (whole cell lysates and IMAC eleutes). 1) hVav 181-375 P182S, S184P, M185T, 2) hVav 181-375 I236T, 3) hVav 181-375 L245F, R246S, 4) hVav 181-375 181-375 T260G, 5) hVav 181-375A264T, N265T, 6) hVav 181-375 R296Q, A299T, 7) hVav 181-375 M351T, 8) hVav 181-375 WT, and 9) mVav 181-375 WT.

Figure 3

Zn²⁺ titrations supplemented to both autoinduction and regular induction conditions for the fermentation of hVav DH-PH-CRD (170-575). A) Autoinduction protocol: 1-4 - whole cell extract in the presence of 0, 100, 500, 1000 μM Zn²⁺; 4-8 – IMAC elutes in the presence of 0, 100, 500, 1000 μM Zn²⁺. In the case of autoinduction protocol ZnCl₂ was added prior to shifting the temperature to 25 °C. B) Regular induction protocol: 1-4 - whole cell extract in the presence of 0, 100, 500, 1000 μM Zn²⁺; 4-8 – IMAC elutes in the presence of 0, 100, 500, 1000 μM Zn²⁺.

Figure 4

In vitro pull-down experiments using GST-Rac1 and (a) hVav-CRD, (b) hVav DH-PH-CRD, and (c) mVav DH. 1) resin control load, 2) resin control wash, 3) resin control GSH elute, 4) experimental load, 5) experimental final wash, and 6) experimental GSH elute.

Figure 5

1D ¹H NMR spectrum of Vav1-CRD at 400 μM in PBS (pH 7.4) with 11 mM TCEP. The spectrum was recorded at 12 °C, with 256 scans and with water suppression by presaturation, on a Bruker Avance 600 spectrometer equipped with a TXI HCN z-gradient probe

Figure 6

(a) Guanine nucleotide exchange assay analysis of “open” and “closed” conformations of hVav DH and “open” conformation of mVav DH at various molar ratios between Vav and Rac1. (b) Guanine nucleotide exchange assay analysis of “open” and “closed” conformations of hVav DH-PH-CRD at various molar ratios between Vav1 and Rac1. Domain boundaries for mVav1 and hVav1 are as indicated in the figure.

Figure 6

(a) Guanine nucleotide exchange assay analysis of “open” and “closed” conformations of hVav DH and “open” conformation of mVav DH at various molar ratios between Vav and Rac1. (b) Guanine nucleotide exchange assay analysis of “open” and “closed” conformations of hVav DH-PH-CRD at various molar ratios between Vav1 and Rac1. Domain boundaries for mVav1 and hVav1 are as indicated in the figure.

Figure 7

Guanine nucleotide exchange assay analysis of Vav1-DH-PH-CRD (190-575) M351T at various molar ratios between Vav1 and Rac1.