Structural and energetic mechanisms of cooperative autoinhibition and activation of Vav1

Abstract

Vav proteins are guanine nucleotide exchange factors (GEFs) for Rho family GTPases. They control processes including T cell activation, phagocytosis, and migration of normal and transformed cells. We report the structure and biophysical and cellular analyses of the five-domain autoinhibitory element of Vav1. The catalytic Dbl homology (DH) domain of Vav1 is controlled by two energetically coupled processes. The DH active site is directly, but weakly, inhibited by a helix from the adjacent Acidic domain. This core interaction is strengthened 10-fold by contacts of the calponin homology (CH) domain with the Acidic, pleckstrin homology, and DH domains. This construction enables efficient, stepwise relief of autoinhibition: initial phosphorylation events disrupt the modulatory CH contacts, facilitating phosphorylation of the inhibitory helix and consequent GEF activation. Our findings illustrate how the opposing requirements of strong suppression of activity and rapid kinetics of activation can be achieved in multidomain systems.

Figure 1

Structural and energetic models of autoinhibition in Vav1. Domains of Vav1 are colored: CH (gold), Ac (red), DH (blue), PH (green), ZF (magenta), SH3 (white), SH2 (white). (A) Domain architecture of Vav1 and thermodynamic model for cooperative inhibition. Solid lines represent direct physical contacts; dashed line represents thermodynamic coupling between core and modulatory equilibria. (B) Schematic representation of autoinhibited CADPZ. Dotted lines outline C-terminal helix of DH domain lying behind PH and CH domains. (C) Ribbon diagram of Vav1 CADPZ. Dashed lines indicate regions not observed in the electron density map. Sidechains of tyrosines 142, 160 and 174 are shown as sticks. DH active site is circled. Boxed areas in left and right panels are expanded in Figures 3A and 3B, respectively. DPZ element overlay from CADPZ and DPZ/Rac complex (2vrw) and selective side chain interactions are shown in Figure S1. Figure prepared using Pymol (Delano, 2002).

Figure 2

Vav1 activity is cooperatively suppressed through energetic coupling of the core and modulatory equilibria. (A) Normalized GEF activity of human Vav1 proteins. Error bars show standard deviation from three independent measurements. NMR data showing that phosphorylation causes dissociation of the CH domain are in Figure S2A. (B) Immobilized GST-Rac(GDP) was used to pull-down the indicated Vav1 proteins, which were separated by SDS-PAGE and visualized by Coomassie Blue staining. M, I, W, E, C represent molecular weight markers, input, final wash, elution and elution from control (GST) beads, respectively. (C) Panc04.03 cells were transfected with a control vector or the indicated full-length Vav1 expression constructs and analyzed for Rac1(GTP) using GST-Pak-PBD. Proteins were resolved by SDS-PAGE and detected by immunoblotting with the indicated antibodies. Transformation assays of the same Vav1 proteins are in Figure S2B–D. (D) Overlaid ¹H/¹³C methyl TROSY spectra of a Leu⁴⁰⁹δ (CH-PH interface) signal in human CADPZ (black), CADPZ_F69A (magenta), CADPZ_F69A/Y174D (cyan), CADPZ_S67D (green), CADPZ_K487E (orange), pCADPZ (red) and ADPZ (blue) proteins. (E) Overlaid ¹H/¹³C methyl TROSY spectra of the Leu³²⁵δ1 resonance (DH-helix interface). Spectra colored as in (D). In (D) and (E), reporter signals are boxed; dashed lines connect endpoints. Spectra showing additional NMR signals are in Figure S2E–I.

Figure 3

Interdomain contacts in Vav1. Domains colored as in Figure 1. (A) Contacts between CH, PH, and DH domains. Residues discussed in the text are shown as sticks. (B) Contacts between CH domain, Ac region and PH domain. Potential hydrogen bonds and ionic interactions are shown as dashed lines. Regions shown in panels A and B are boxed in black and red, respectively, in Figure 1C.

Figure 4

Interdomain cooperativity in Vav1. (A) Overlaid ¹H/¹³C methyl TROSY spectra of murine A_sD and CADPZ proteins (left and right panels, respectively), showing Leu¹⁸⁰δ1 and Leu³²⁵ δ1 signals (top and bottom panels, respectively). Colors are: WT (black) and K208A (blue), K208S (yellow), K208G (magenta) mutants and pA_sD or CADPZ_Y174D (red). Relaxation dispersion data for A_sD, AD, CAD, CADPZ and methyl TROSY spectra showing additional NMR signals are in Figure S3. Dotted lines connect resonances from phosphorylated and non-phosphorylated proteins. (B) Ratio of open and closed populations (p_o/p_c, average ± standard deviation from 5–8 resonances) of murine A_sD (red bars) and CADPZ (black bars) proteins. (C) Normalized GEF activity of murine A_sD (red; previously shown in (Li et al., 2008) and reproduced here by permission from Macmillan Publishers Ltd) and CADPZ proteins (black). Error bars show standard deviation from 3 independent measurements.

Figure 5

Inhibitory effects of the CH domain require an intact Ac element. Relative GEF activity in titrations of CH proteins into ADPZ proteins: CH+ADPZ (red circles), CA+A_sDPZ (blue squares). Activities were normalized to those of free ADPZ or A_sDPZ, respectively. Error bars represent standard deviations from three independent measurements. Cartoon on the right depicts the corresponding titration reaction. Arrow indicates the position where the Ac element is cut. NMR titrations and comparison of CH titration into ADPZ with control experiments are shown in Figure S4.

Figure 6

Initial phosphorylation of Tyr¹⁴² and Tyr¹⁶⁰ can provide a route to full Vav1 activation. (A) Normalized GEF activity of human Vav1 proteins. Error bars represent standard deviations from three independent measurements. (B) Overlaid ¹H/¹³C methyl TROSY spectra of the Leu³²⁵δ1 signal (boxed) in human CADPZ (black), ADPZ (blue) and CADPZ_Y142D (magenta), CADPZ_Y174D (green), CADPZ_Y142D/Y160D (gold), pCADPZ (red) proteins. Spectra showing additional NMR signals and relaxation dispersion analysis of CADPZ are in Figure S5. (C) HPLC analysis (with scintillation detection) of tryptic digests of CADPZ (black) and CADPZ_Y142D/Y160D (red) after phosphorylation with [γ-³²P] ATP for 7.5 minutes. Peaks corresponding to pTyr¹⁴²-, pTyr¹⁶⁰- and pTyr¹⁷⁴- containing peptides are indicated. At complete phosphorylation, the peak corresponding to pY174 has virtually identical amplitude for the two proteins (not shown). HPLC analysis of tryptic digests of CADPZ and CADPZ_D39N after phosphorylation with [γ-³²P] ATP are in Figure S5.