Crystal Structure of the GTPase-activating Protein-related Domain from IQGAP1

Vinodh B Kurella,Jessica M Richard,Courtney L Parke,Louis F Lecour,Henry D Bellamy,David K Worthylake

doi:10.1074/jbc.m808974200

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.

Highlights

To whom correspondence should be addressedThe IQ repeats have been shown to mediate binding to calmodulin and calmodulin-like proteins (e.g. S100, myosin essential light chain), whereas the GTPase-activating protein (GAP)-related domain (GRD) does not appear to bind to Ras but instead is necessary for binding to the Rho family GTPases Cdc and Rac, primarily in their active forms (9 –11)
Whereas the intrinsic rate of GTP hydrolysis on Rho proteins is faster than Ras, this rate can be stimulated by interaction with a RhoGAP
The GAP-related domain (GRD) structure is quite similar to the GTPase-activating protein (GAP) domains of p120, neurofibromin, and SynGAP; unlike those domains, the GRD possesses a conserved threonine in place of the catalytic arginine finger and has a 31-residue insertion that projects from one end of the molecule

Summary

To whom correspondence should be addressed

The IQ repeats have been shown to mediate binding to calmodulin and calmodulin-like proteins (e.g. S100, myosin essential light chain), whereas the GAP-related domain (GRD) does not appear to bind to Ras but instead is necessary for binding to the Rho family GTPases Cdc and Rac, primarily in their active forms (9 –11). Instead of accelerating hydrolysis of GTP, IQGAP1 preserves the activated states of Cdc and Rac to the extent that overexpression of IQGAP1 in cells increases the levels of active GTPase [12]. It appears that IQGAP1 is an effector of Cdc and Rac and preserves their activated states by tightly binding to the GTPases and stabilizing them in a conformation not conducive to GTP hydrolysis. Sequence conservation mapped to the surface of the GRD indicates that the surface with the highest degree of conservation overlaps with the surface that makes contacts to Cdc in the model

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION