The Structural Basis for Cdc42-Induced Dimerization of IQGAPs

Abstract

In signaling, Rho-family GTPases bind effector proteins and alter their behavior. Here we present the crystal structure of Cdc42·GTP bound to the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP2. Four molecules of Cdc42 are bound to two GRD molecules, which bind each other in a parallel dimer. Two Cdc42s bind very similarly to the Ras/RasGAP interaction, while the other two bind primarily to "extra domain" sequences from both GRDs, tying the GRDs together. Calorimetry confirms two-site binding of Cdc42·GTP for the GRDs of both IQGAP2 and IQGAP1. Mutation of important extra domain residues reduces binding to single-site and abrogates Cdc42 binding to a much larger IQGAP1 fragment. Importantly, Rac1·GTP displays only single-site binding to the GRDs, indicating that only Cdc42 promotes IQGAP dimerization. The structure identifies an unexpected role for Cdc42 in protein dimerization, thus expanding the repertoire of interactions of Ras family proteins with their targets.

Figure 1

IQGAP1 schematic. CH; calponin homology domain, REPEATS; 6 ~50 amino acid sequence repeats, WW; tryptophan-containing polyproline-binding domain, IQ; 4 isoleucine- and glutamine-containing motifs, GRD; RasGAP-related domain, RGCT; RasGAP C-terminal domain found in IQGAPs. Underlined in green is the region previously shown to be important for mediating IQGAP1 dimerization (residues 763-863). Underlined in magenta are the equivalent Ex domain residues that participate in the GRD2-GRD2 interface in the crystal structure (IQGAP2 residues within 875-914 and 1204-1246). Indicated are the amino acids used for the GRD constructs. Note, all 3 human IQGAPs have a similar domain composition and layout.

Figure 2

The crystal asymmetric unit. Two IQGAP2 GRDs (brown, yellow) are bound to each other and each binds to two molecules of Cdc42 (cyan, green). The conformation-variable “switches” of Cdc42 are in light red and the 31 GRD residues with no structural equivalent in RasGAP, neurofibromin or SynGAP are colored magenta. The cyan Cdc42 molecules A and B (cyan letters) bind primarily to residues belonging to the Ex sub-domain of GRD molecules E and F, respectively (yellow and brown letters). Cdc42 molecules C and D (green letters) are binding in a mode very similar to the Ras/RasGAP interaction to GRD molecules E and F, respectively. Panels A and B are in the same orientation. Panel C has been rotated exactly 90° about the horizontal. See also Supplemental Figures S1 and S2.

Figure 3

Comparison of the IQGAP1 and IQGAP2 GRDs. A, GRD2 Molecule E with Ex domain residues (875-934 and 1204-1246) and central domain residues (935-1203) in blue and yellow, respectively. Residues with no structural equivalent in RasGAPs are in magenta. B, The unbound GRD1 structure (Kurella et al., 2009)with Ex domain residues (962-1021 and 1291-1333) and central domain residues (1022-1290) colored similarly. Note the large structural differences between the two 80% identical Ex domains including the β-strand in GRD2 (black arrow). The two GRDs were aligned for the figure using LSQKAB (CCP4, 1994) and residues 939-1199 (GRD2 numbering). The r.m.s.d. for the 261 Cα atoms is 1.75Å.

Figure 4

Common contacts for Cdc42 molecules in RasGAP- and Ex-binding modes. A, RasGAP-mode Cdc42 molecule with GTP and magnesium (sticks and blue sphere) surrounded by the two switch regions (salmon). B, Ex-mode Cdc42 with switches, GTP, and magnesium rendered similarly. Nearly all Cdc42 residues in black lettering lose >20Ų of solvent accessible surface area upon binding to the GRD2. GRD2 residues in red are van der Waals’ contacts, and residues in green and blue denote hydrogen-bonds and salt bridges, respectively. Green and black asterisks indicate that the GRD2 or the GTPase sidechain, respectively, is used in the hydrogen bond. Green residues with no asterisk are involved in mainchain-to-mainchain hydrogen bonds. Underlined GRD2 residues contacting the cyan molecules are from the “opposite” GRD2 (e.g. Cdc42 molecule A contacted by GRD2 molecule F). See also Supplemental Figures S3 and S4.

Figure 5

Stereo view of important Cdc42 “switch” interactions in the RasGAP- and Ex-binding modes. A, RasGAP-mode switch 1 and switch 2 (salmon) hydrogen bonds and salt bridges (dashed lines) to the GRD2 (yellow) as defined by PDBePISA (see Methods). Gln⁶¹ has been modeled in the same conformation that is found in PDB ID 1GRN (Nassar et al., 1998). B, Key Ex-mode switch 1 interactions with the GRD2 are represented by dashed lines. The bound nucleotide is shown in stick representation with carbon atoms in light grey. Note the direct contacts to the nucleotide atoms O2 and O3 of the sugar and N2 of the base. See also Supplemental Figures S3 and S4.

Figure 6

Isothermal titration calorimetry indicates two-site binding by Cdc42 for both GRD1 and GRD2. Experiments were performed at 30°C and integrated heats were fitted to the “TwoSites” model using Origin 7 software. All experiments have Cdc42 (Q61L; residues 1-177) in the syringe and the wild-type GRD protein in the cell. A, GRD1 estimated dissociation constants are: K_d1 = 9.9 × 10⁻⁸ M, K_d2 = 2.6 × 10⁻⁶ M. B, GRD2 estimated dissociation constants are: K_d1 = 6.8 × 10⁻⁷ M, K_d2 = 6.0 × 10⁻⁶ M. See also Supplemental Figures S5, S6 and S7.