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. 2005 Apr 19;102(16):5685-90.
doi: 10.1073/pnas.0406472102. Epub 2005 Apr 8.

The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott-Aldrich syndrome protein

Daisy W Leung  1 , Michael K Rosen
Affiliations

The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott-Aldrich syndrome protein

Daisy W Leung et al. Proc Natl Acad Sci U S A. .

Abstract

The GTP/GDP nucleotide switch in Ras superfamily GTPases generally involves differential affinity toward downstream effectors, with the GTP-bound state having a higher affinity for effector than the GDP-bound state. We have developed a quantitative model of allosteric regulation of the Wiskott-Aldrich syndrome protein (WASP) by the Rho GTPase Cdc42 to better understand how GTPase binding is coupled to effector activation. The model accurately predicts WASP affinity for Cdc42, activity toward Arp2/3 complex, and activation by Cdc42 as functions of a two-state allosteric equilibrium in WASP. The ratio of GTPase affinities for the inactive and active states of WASP is appreciably larger for Cdc42-GTP than for Cdc42-GDP. The greater ability to distinguish between the two states of WASP makes Cdc42-GTP a full WASP agonist, whereas Cdc42-GDP is only a partial agonist. Thus, the nucleotide switch controls not only the affinity of Cdc42 for its effector but also the efficiency of coupling between the Cdc42-binding and allosteric equilibria in WASP. This effect can ensure high fidelity and specificity in Cdc42 signaling in crowded membrane environments.

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Figures

Fig. 1.
Fig. 1.
Domain organization of WASP constructs used in this study. EVH1, Ena/Vasp homology 1; B, basic region; P, proline-rich region; V, verprolin homology region; C, central hydrophobic domain; A, acidic region; GGSGGS, linker. Color coding matches that of the autoinhibited WASP structure in Fig. 3.
Fig. 2.
Fig. 2.
A two-state allosteric model for WASP. WASP exists in equilibrium between an inactive, folded autoinhibited state and an active, predominantly unfolded state. Cdc42 biases this equilibrium toward activation because of its higher affinity for the active state. WASPi and WASPa represent inactive and active states of WASP, respectively.
Fig. 3.
Fig. 3.
Mutations generated in this study. The minimal autoinhibited WASP structure (Protein Data Bank ID code 1EJ5) (9) consists of the GBD (residues 242–310; yellow and cyan) connected to the C domain helix (residues 461–492; red) by a GGSGGS linker. Elements of the GBD that contact Cdc42 directly in the Cdc42–GBD structure (Fig. 8A) (11) are yellow. Side chains of residues mutated in the background of the GBD–VCA construct are purple and are located in the GBD outside of the Cdc42 binding interface.
Fig. 4.
Fig. 4.
Affinity of WASP proteins as a function of stability. KD values for Cdc42–GMPPNP are shown as black circles. The black line indicates the log fit of the data to Eq. 5.
Fig. 5.
Fig. 5.
Activity of WASP proteins as a function of stability. (A) Pyrene-actin polymerization assays of 500 nM WASP proteins in the presence of 10 nM Arp2/3 complex (except “actin only” curve) and 4 μM actin (6% pyrene-labeled). (B) Actin polymerization assays comparing GBD–VCA mutant constructs with BGBD–VCA mutant constructs (indicated by a B-prefix). (C) Fractional activity (relative to VCA) of WASP proteins in the pyrene-actin polymerization assay. GBD–VCA and BGBD–VCA (wild-type and mutant proteins) are indicated by circles and triangles, respectively. The fractional activities predicted by the model in the absence of activator (Eq. 11 in Supporting Methods), in the presence of Cdc42–GDP (Eq. 12 in Supporting Methods), and in the presence of Cdc42–GTP are shown as black solid, green dashed, and blue dotted lines, respectively. The C values used for Cdc42–GMPPNP and Cdc42–GDP are 1.1 × 10–2 and 5.4 × 10–2, respectively.
Fig. 6.
Fig. 6.
WASP activity in the presence of activators. (A) Fractional activity of different WASP constructs with increasing fractional saturation by Cdc42–GMPPNP. Lines show linear fits of the data for each protein. (B) Actin polymerizations assays of M307A were performed with increasing saturation by Cdc42–GMPPNP (blue) or Cdc42–GDP (green). Data points were fit to a linear slope and extrapolated to 100% saturation.
Fig. 7.
Fig. 7.
1H/15N TROSY HSQC spectra of 2H/15N GBD–VCA. (A) GBD–VCA (100 μM). (B) GBD–VCA (100 μM) in the presence of 500 μM Cdc42–GMPPNP (99% saturation). (C) GBD–VCA (100 μM) in the presence of 750 μM Cdc42–GDP (90% saturation). Peaks identified in A (red squares) and superimposed on C are GBD–VCA residues, including the CRIB motif, which broaden severely upon binding to Cdc42–GDP. Additional peaks identified in B and C (blue circles) are new downfield shifted peaks that appear upon binding to Cdc42–GTP (11) and Cdc42–GDP, respectively.
Fig. 8.
Fig. 8.
Binding of WASP to Cdc42–GTP and Cdc42–GDP. (A) Structure of a truncated WASP GBD (residues 230–277; yellow) bound to Cdc42–GTP (residues 1–178; purple) (Protein Data Bank ID code 1CEE) (11). The WASP CRIB motif is highlighted in orange. (B) The structure of Cdc42–GDP (purple; Protein Data Bank ID code 1DOA) (32). Residues whose 1H/15N TROSY HSQC resonances decrease in intensity by >60% or shift by >0.033 ppm when 50 μM GBD–VCA is added to 100 μM 2H/15N Cdc42–GDP are colored yellow. Differences in 1H and 15N chemical shifts were determined by using the equation Δδ = [(Δ1H)2 + (Δ15N*0.15)2]1/2. (C) Normalized ratios of 1H/15N Cdc42–GDP peak intensities in the presence of GBD–VCA relative to 1H/15N Cdc42–GDP peak intensities alone. Cdc42–GDP resonances that were absent from the spectra are indicated by an intensity ratio of 0. The secondary structural elements of Cdc42–GDP are indicated below.
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