Crystal structure of the GTPase-activating protein-related domain from IQGAP1

Abstract

IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic "arginine finger" seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099-1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42.GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.

FIGURE 1.

Experimental electron density. Density-modified experimental phases and λ₁ amplitudes (all |F| > 0) were used to calculate this map in the resolution range of 25-2.4 Å. The map is contoured at 1.4 σ. In the lower right of the picture are Tyr¹¹⁹³ and Arg¹¹⁹⁴.

FIGURE 2.

The IQGAP1 GRD and GAP-334 have very similar tertiary structures. The GRD (left) and GAP-334 (Protein Data Bank code 1WER; right) were superimposed using combinatorial extension (29) to align the coordinates for this figure (root mean square deviation = 3.3 Å for 312 Cα atoms). The cartoon has been colored orange and yellow for the Ex domains of the GRD and GAP-334, respectively. The central domains are colored blue or light blue, and the 31-residue insertion characteristic of IQGAP GRDs is colored green.3₁₀ helices are shown in dark gray, and a five-residue π helix containing the ¹¹⁹²YYR¹¹⁹⁴ motif in the GRD is shown in dark blue. Thr¹⁰⁴⁶ of the GRD and Arg⁷⁸⁹ of GAP-334 are shown as stick representations in magenta.

FIGURE 3.

Structure-based sequence alignment of the GRD and GAP-334. The three-dimensional coordinates of the GRD and GAP-334 (Protein Data Bank code 1WER) were superimposed using combinatorial extension (29) to produce this structure-based sequence alignment. Residues at the termini of the GRD and GAP-334 that were disordered or did not have a structural equivalent are shown as blue or gray letters, respectively. Secondary structure was assigned using DSSP (46) and colored as in Fig. 2. Residues that are invariant in an alignment of 55 IQGAP GRD homologs are underlined, and residues that contact Cdc42 in the model or Ras in the Ras/GAP-334 complex (Protein Data Bank code 1WQ1) are colored red. The GRD secondary structure colored green (residues 1099–1129) has no equivalent in GAP-334.

FIGURE 4.

Interactions mediated by Arg¹¹⁹⁴ of the YYR motif characteristic of IQGAP proteins. The aligned coordinates of the GRD (left) and GAP-334 (Protein Data Bank code 1WER; right) were used to make this figure. Instead of containing an FLR signature characteristic of RasGAPs, IQGAP GAP-related domains possess a YYR motif. Whereas Arg¹¹⁹⁴ makes just one hydrogen bond to stabilize helix α2a_c of the GRD, Arg⁷⁸⁹ interacts with three backbone oxygen atoms of the finger loop of GAP-334. α2a_c of the GRD is further stabilized by a hydrogen bond between the Thr¹⁰⁴⁶ side chain and the backbone oxygen of Gln¹⁰⁴².

FIGURE 5.

Surface representation of sequence conservation and model contacts. Left panel, a surface representation of the GRD, oriented approximately the same as in the lower left of Fig. 2, has been colored to indicate residues that are at least 90% (red) or 100% (magenta) identical among 55 metazoan IQGAP GRDs. Right panel, residues that lose >10 Ų solvent-accessible surface area in the Cdc42/GRD model have been colored blue. In general, the highest degree of sequence conservation includes residues that contact Cdc42. Note the high degree of sequence conservation in the 31 residue insertion (arrow).

FIGURE 6.

Close-up of the model active site. Thr¹⁰⁴⁶ of the Cdc42/GRD model (see “Experimental Procedures”) impinges on the phosphate-binding loop (P-Loop) resulting in a ∼1.8 Å displacement (red arrow) as compared with the energy-minimized model of isolated Cdc42·GTP (transparent representation). The shift in the P-loop displaces the nucleotide and Mg²⁺. Tyr¹¹⁹³ and Arg¹¹⁹⁴ bracket Gln⁶¹ of Cdc42, and both the glutamine and tyrosine hydrogen bond with the nucleophilic Wat²³⁰ (distances of 3.0 and 2.8 Å, respectively). Not shown are the hydrogen bonds from the Tyr¹¹⁹³ hydroxyl to 35O of Cdc42 (distance of 2.8 Å) and Gln⁶¹ NE2 to 1045O of the GRD (distance of 3.1 Å).

FIGURE 7.

The isolated IQGAP1 GRD binds GTP-bound Cdc42. GRD protein and Cdc42(Q61L)·GTP were dialyzed for 24 h against 2 × 2 liters of: 50 mm Tris, pH 8.0, 100 mm NaCl, 100 mm KCl, 5% glycerol, 5 mm MgCl₂, and 2 mm β-mercaptoethanol at 4 °C. 120 μm GRD was placed into the calorimeter cell, and 10 μl of 680 μm Cdc42 was injected at 210-s intervals. The experiment was conducted at 20 °C using a MicroCal™ VP-ITC calorimeter. The raw data (top) was converted to integrated heats (bottom), and Origin 7.0 software (MicroCal™) was used to curve fit using a model of “one site” binding. The binding constant corresponds to K_d ≈ 1.3 μm. The binding is endothermic, indicating an entropically driven binding reaction.