Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1

Abstract

The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 A resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1's ability to contact a range of Rho GTPases using a novel protein-protein interaction network.

Figure 1

Figure 1A. Domain architecture of the Vav family of guanine nucleotide exchange factors. Vav1 consists of a calponin 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 cysteine rich domain (CRD, 516–565), a proline rich region (607–610), and an SH3-SH2-SH3 domain (612–844). Figure 1B. Overall structure of the Vav-1 DH-PH-CRD in complex with the Rac1 RhoGTPase solved to a resolution of 2.6 Å and a free R factor of 29%. The Vav1 DH domain is depicted in blue, the PH domain is depicted in cyan, and the CRD is shown in magenta, with grey spheres representing coordinating zinc ions. Rac1 is shown in green, with the Switch I and Switch II regions highlighted in red.

Figure 1

Figure 1A. Domain architecture of the Vav family of guanine nucleotide exchange factors. Vav1 consists of a calponin 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 cysteine rich domain (CRD, 516–565), a proline rich region (607–610), and an SH3-SH2-SH3 domain (612–844). Figure 1B. Overall structure of the Vav-1 DH-PH-CRD in complex with the Rac1 RhoGTPase solved to a resolution of 2.6 Å and a free R factor of 29%. The Vav1 DH domain is depicted in blue, the PH domain is depicted in cyan, and the CRD is shown in magenta, with grey spheres representing coordinating zinc ions. Rac1 is shown in green, with the Switch I and Switch II regions highlighted in red.

Figure 2

Figure 2A. Complete interlock network between the Vav1 DH domain, PH domain, and CRD. The DH domain is depicted in blue, the PH domain in cyan, and the CRD in magenta. Critical interactions between the domains are shown with dotted black lines. Figure 2B. Superposition of the Vav1 cysteine rich domain; magenta) on the Raf1 cysteine rich domain (grey) previously described. The overall structures are similar, with both similarly coordinating two zinc ions, depicted as spheres.

Figure 2

Figure 2A. Complete interlock network between the Vav1 DH domain, PH domain, and CRD. The DH domain is depicted in blue, the PH domain in cyan, and the CRD in magenta. Critical interactions between the domains are shown with dotted black lines. Figure 2B. Superposition of the Vav1 cysteine rich domain; magenta) on the Raf1 cysteine rich domain (grey) previously described. The overall structures are similar, with both similarly coordinating two zinc ions, depicted as spheres.

Figure 3

SAXS analysis of Vav1-DH-PH-CRD/Rac1 structure. The scattering profiles are depicted for the experimental data, shown in black, for the crystal structure, in red, for both molecules present in asymmetric unit, green dashed line, and for the model proposed by Llorca et al. that contains a different orientation of the CRD and PH domains, pink dashed line. The crystal structure is in close agreement with the experimental scattering profile.

Figure 4

a) The PH/CRD region (blue) of the Vav1-DH-PH-CRD/Rac1 complex rotated 360° at 10° increments about the base of the C-terminal DH connecting helix. b) A plot of the PH/CRD rotation angle vs χ² fit of the calculated scattering curve to the experimental scattering curve. This plot indicates that the crystal structure, and angles of PH/CRD rotation close to this structure, best agree with the experimental data (Fig. 4b). c) A plot of the theoretical SAXS curve of four alternate conformations, the crystal structure, rotation of the PH/CRD region by 90°, by 180° (as in Fig 3) and by 270°, plotted with the experimental SAXS data. The crystal structure is the optimal fit of these four conformations. d) The ab initio SAXS shape, generated by averaging 10 independent runs of GASBOR, overlaid on the Vav1-DH-PH-CRD/Rac1 crystal structure. The ab initio SAXS shape and crystal structure are in agreement, further supporting the closed conformation of the crystal structure rather than an extended conformation indicated by EM analysis.

Figure 5

Guanine nucleotide exchange assay analysis of representative Rac1 Switch II mutants. Exchange reactions were carried out in triplicates as described in the Materials and Methods. Exchange rates are calculated as percentages of the exchange rate of wild type Rac1.

Figure 6

Figure 6A. Interlock system between the Vav1 CRD and Rac1 Switch II region with the α11 C-terminal helix of the Vav1 DH domain. The CRD is shown in magenta, the DH domain in blue, and the Rac1 in green. Polar interactions are depicted in grey, while hydrogen bonds in black. The series of interactions at this interface is predicted to stabilize this flexible helix for an integrated interaction with a particular RhoGTPase. Figure 6B. σ-A weighted 2|F_obs | − |F_calc| electron density at 2.6 Å resolution, contoured at 1.5 σ for the interlock system between the Vav1 CRD and Rac1 Switch II region with the α11 C-terminal helix of the Vav1 DH domain. Figure 6C. The interaction network between the Rac1 Switch I region and the Vav1 DH domain. The DH domain is shown in blue, and Rac1 in green. Hydrogen bonds are depicted as dotted black lines. The spatial orientation of Trp-56 is also shown, although this residue forms no interactions with residues from Vav1.

Figure 6

Figure 6A. Interlock system between the Vav1 CRD and Rac1 Switch II region with the α11 C-terminal helix of the Vav1 DH domain. The CRD is shown in magenta, the DH domain in blue, and the Rac1 in green. Polar interactions are depicted in grey, while hydrogen bonds in black. The series of interactions at this interface is predicted to stabilize this flexible helix for an integrated interaction with a particular RhoGTPase. Figure 6B. σ-A weighted 2|F_obs | − |F_calc| electron density at 2.6 Å resolution, contoured at 1.5 σ for the interlock system between the Vav1 CRD and Rac1 Switch II region with the α11 C-terminal helix of the Vav1 DH domain. Figure 6C. The interaction network between the Rac1 Switch I region and the Vav1 DH domain. The DH domain is shown in blue, and Rac1 in green. Hydrogen bonds are depicted as dotted black lines. The spatial orientation of Trp-56 is also shown, although this residue forms no interactions with residues from Vav1.

Figure 6

Figure 6A. Interlock system between the Vav1 CRD and Rac1 Switch II region with the α11 C-terminal helix of the Vav1 DH domain. The CRD is shown in magenta, the DH domain in blue, and the Rac1 in green. Polar interactions are depicted in grey, while hydrogen bonds in black. The series of interactions at this interface is predicted to stabilize this flexible helix for an integrated interaction with a particular RhoGTPase. Figure 6B. σ-A weighted 2|F_obs | − |F_calc| electron density at 2.6 Å resolution, contoured at 1.5 σ for the interlock system between the Vav1 CRD and Rac1 Switch II region with the α11 C-terminal helix of the Vav1 DH domain. Figure 6C. The interaction network between the Rac1 Switch I region and the Vav1 DH domain. The DH domain is shown in blue, and Rac1 in green. Hydrogen bonds are depicted as dotted black lines. The spatial orientation of Trp-56 is also shown, although this residue forms no interactions with residues from Vav1.

Figure 7

Endogenous Rac1 activation induced by exogenous expressed wild type Vav1 and Vav1 mutants in HEK293 cells. A) The expression levels of wild type Vav1 and Vav1 mutants in HEK293 cells were detected by Vav1 antibody and oncogenic Vav-1 was detected by HA antibody. B) The expression levels of endogenous Rac1 in HEK 293 cells expressing wild type Vav1 and Vav1 mutants were detected by Rac1 antibody. C) Active Rac1 in HEK293 cells expressing wild type Vav1 and Vav1 mutant was detected by Pull-down assay (described in methods and materials).

Figure 8

Disruption of the β2/β3 region interface in Vav1/Rac1 structure. Comparison of the Tiam1/Rac β2/β3 region (left) and the Vav/Rac β2/β3 region (right). Tiam-1 (cyan) forms an integrated network with the B2/B3 region of Rac1 (grey), in addition to a hydrogen bond between Trp-56 of Rac1 and His-1178 of Tiam1. Vav1 (blue) forms only one hydrogen bond in this region, between Ser-41 of the B2/B3 of Rac1 (green) and Arg-319 of the DH domain of Vav1. In addition, Trp-56 of Rac1 does not form any interaction with the DH domain of Vav1, polar or hydrophobic.

Figure 9

Guanine nucleotide exchange assay analysis of Rac1 Trp 56 and β2/β3 region mutants. Exchange reactions were carried out in triplicates as described in the Materials and Methods. A) Wild type Rac1; B) Rac1 S41A; C) Rac1 N43A; D) Rac1 W56F. Molar ratio of GEF/RhoGTPase were adjusted to generate similar dynamic range (Vav-DH-PH-CRD 1:8; Tiam DH-PH 2:1; Vav-DH 8:1).