Crystal Structure of the GTPase-activating Protein-related Domain from
IQGAP1 |
| |
Authors: | Vinodh B Kurella Jessica M Richard Courtney L Parke Louis F LeCour Jr Henry D Bellamy and David K Worthylake |
| |
Institution: | ‡Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112 and the §Center for Advanced Microstructures and Devices, Louisiana State University, Baton Rouge, Louisiana 70806 |
| |
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.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 (~1.2 × 10–4
s–1), but this rate of hydrolysis can be enhanced
~105-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 glutamine-rich motifs (IQ
repeats) and a region with sequence homology to the Ras-specific GAP domains
of p120RasGAP, NF1, and SynGAP
(2–4,
8). Subsequently, two homologs,
IQGAP2 and IQGAP3, have been discovered. 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 Cdc42 and Rac1, primarily in their active forms
(9–11).
However, instead of accelerating hydrolysis of GTP, IQGAP1 preserves the
activated states of Cdc42 and Rac1 to the extent that overexpression of IQGAP1
in cells increases the levels of active GTPase
(12). Because IQGAP1
expression increases the level of activated Cdc42, initially there was some
confusion as to whether the protein might not represent a novel guanine
nucleotide exchange factor. However it now appears that IQGAP1 is an effector
of Cdc42 and Rac1 and preserves their activated states by tightly binding to
the GTPases and stabilizing them in a conformation not conducive to GTP
hydrolysis. IQGAP1 appears to be such an important effector for Cdc42 that
abrogation of binding to IQGAP1 not only reduces the levels of active Cdc42,
it also reduces membrane-localized Cdc42 and the cellular response to
bradykinin (12).A growing body of evidence implicates IQGAP1 in carcinogenesis. Expression
of IQGAP1 increases during the transition from a minimally to a highly
metastastic form of melanoma, and IQGAP1 has been found to be overexpressed in
ovarian, breast, lung, and colorectal cancers
(13–17).
In vitro, overexpressed IQGAP1 enhances cell motility and
invasiveness in a process that requires Cdc42 and Rac
(18). β-Catenin is one of
the many binding partners of IQGAP1 identified to date. IQGAP1 has been shown
to bind to β-catenin and interfere with β-catenin binding to
α-catenin, an interaction necessary for stable cell-cell adhesion
(19). Another study found that
IQGAP2 knock-out mice overexpress IQGAP1 and developage-dependent liver cancer
and apoptosis (20).To better understand how a protein domain homologous to others that
accelerate GTP hydrolysis can function as an effector and preserve the
GTP-bound state, we have determined the x-ray structure of the IQGAP1 GRD.
Despite low sequence identity, the GRD structure is quite similar to the GAP
domains of p120, neurofibromin, and SynGAP; however, 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.
Using the coordinates of Ras·GDP·AlF3 in complex with
the GAP domain of p120, we built a model of Cdc42·GTP bound to the GRD.
The model indicates that a steric clash between the conserved
Thr1046 and the phosphate-binding loop of Cdc42 and other subtle
changes within the active site would likely preclude nucleotide 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 Cdc42 in the model. |
| |
Keywords: | |
|
|