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 Cdc42⅐GTP 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.The small GTPase Ras functions as a binary switch in cell signaling processes. When bound to GTP, Ras is able to interact with effector proteins, including Raf kinase, and alter their activities. Ras signaling is terminated when bound GTP is hydrolyzed to GDP and inorganic phosphate. The basal rate of GTP hydrolysis on Ras is quite slow (ϳ1.2 ϫ 10 Ϫ4 s Ϫ1), but this rate of hydrolysis can be enhanced ϳ10 5 -fold by interaction with a GTPase-activating protein (GAP) 2 (1). Several RasGAPs have been identified to date including p120 RasGAP and neurofibromin (NF1). The Rho family of Ras-related small GTPases also function as binary switches in cell signaling processes. Whereas the intrinsic rate of GTP hydrolysis on Rho proteins is faster than Ras, this rate can also be stimulated by interaction with a RhoGAP. Examination of the structures of the GAP domains of p120RasGAP (2), neurofibromin (3), SynGAP (4), and the GAP domains from the RhoGAPs p50 RhoGAP and the Bcr homology domain of phosphatidylinositol 3-kinase (5, 6) indicates that although ostensibly different, these all-helical domains are structurally related (7).IQGAP1 was discovered by chance during an attempt to isolate novel matrix metalloproteinases (8). Analysis reveals that the protein contains several discrete domains and motifs including a region containing four isoleucine-and glutaminerich motifs (IQ repeats) and a region with sequence homology to the Ras-specific GAP domains of p120RasGAP, NF1, and

IQGAPs as Key Regulators of Actin-cytoskeleton Dynamics

“…The name "IQGAP" originates as a result of this domain organization, but no report has convincingly demonstrated its GAP activity toward any small GTPases, which is in agreement with the finding that the GAP domain of IQGAPs lacks the arginine finger that is critical for GAP activity (Kurella et al, 2009). Subsequent studies revealed IQGAP1 to be an effector of Rac1 and Cdc42, and the GRD domain in IQGAP1 was later shown to stabilize Rac1 and Cdc42 in the GTP-bound active form (Noritake et al, 2005;White et al, 2012).…”

IQGAPs choreograph cellular signaling from the membrane to the nucleus

“…The GRD illustrates other unique properties of IQGAPs. Though similar in sequence to GAPs, the IQGAP GRD has an arginine replaced by threonine at a conserved catalytic residue found in GAPs [107]. This is a key feature to bind to and stabilize small G-proteins in their GTP bound form.…”

Effect of Cdc42 domains on filopodia sensing, cell orientation, and haptotaxis

“…The surrounding region, 1045-1195, was investigated because it included residues that are also implicated in Cdc42 binding. Thr1046 is needed to inhibit the GTPase [23], whereas Tyr1192 and Arg1194 were predicted to play a role in Cdc42 binding [39]. For the Rac ligand outside the CBD, nine conformations forming multiple H-bonds were predicted.…”

“…Chimera software is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco. The crystallographic structure of the GAP-related domain of IQGAP1, 3FAY [23], was obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) database at bwww.rcsb.orgN [24]. The target, 3FAY, and ligands were submitted for blind docking using SwissDock software from Swiss Institute of Bioinformatics [25].…”