Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP

The Rho-related small GTP-binding protein Cdc42 has a low intrinsic GTPase activity that is significantly enhanced by its specific GTPase-activating protein, Cdc42GAP. In this report, we present the tertiary structure for the aluminum fluoride-promoted complex between Cdc42 and a catalytically active domain of Cdc42GAP as well as the complex between Cdc42 and the catalytically compromised Cdc42GAP(R305A) mutant. These structures, which mimic the transition state for the GTP hydrolytic reaction, show the presence of an AlF3 molecule, as was seen for the corresponding Ras–p120RasGAP complex, but in contrast to what has been reported for the Rho–Cdc42GAP complex or for heterotrimeric G protein α subunits, where AlF4 – was observed. The Cdc42GAP stabilizes both the switch I and switch II domains of Cdc42 and contributes a highly conserved arginine (Arg 305) to the active site. Comparison of the structures for the wild type and mutant Cdc42GAP complexes provides important insights into the GAP-catalyzed GTP hydrolytic reaction.

[1]  S. Bagrodia,et al.  Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. , 1994, The Journal of biological chemistry.

[2]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[3]  R. Goody,et al.  Formation of a Transition-State Analog of the Ras GTPase Reaction by Ras·GDP, Tetrafluoroaluminate, and GTPase-Activating Proteins , 1996, Science.

[4]  S. Sprang,et al.  Structure of RGS4 Bound to AlF4 −-Activated Giα1: Stabilization of the Transition State for GTP Hydrolysis , 1997, Cell.

[5]  S. Sprang.  GAP into the Breach , 1997, Science.

[6]  S. Smerdon,et al.  Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP , 1997, Nature.

[7]  Wolfgang Kabsch,et al.  Evaluation of Single-Crystal X-ray Diffraction Data from a Position-Sensitive Detector , 1988 .

[8]  R. Lin,et al.  Biochemical Studies of the Mechanism of Action of the Cdc42-GTPase-activating Protein* , 1998, The Journal of Biological Chemistry.

[9]  D. Herschlag,et al.  Ras-catalyzed hydrolysis of GTP: a new perspective from model studies. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  H. Hamm,et al.  GTPase mechanism of Gproteins from the 1.7-Å crystal structure of transducin α - GDP AIF−4 , 1994, Nature.

[11]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[12]  W. Minor,et al.  Crystal structure of RhoA–GDP and its functional implications , 1997, Nature Structural Biology.

[13]  Heidi E. Hamm,et al.  The 2.2 Å crystal structure of transducin-α complexed with GTPγS , 1993, Nature.

[14]  W A Hendrickson,et al.  Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three‐dimensional structure. , 1990, The EMBO journal.

[15]  M. Hirshberg,et al.  The crystal structure of human rac1, a member of the rho-family complexed with a GTP analogue , 1997, Nature Structural Biology.

[16]  A. Hall,et al.  Characterization of rhoGAP. A GTPase-activating protein for rho-related small GTPases. , 1994, The Journal of biological chemistry.

[17]  W. Kabsch,et al.  The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. , 1997, Science.

[18]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[19]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[20]  A. Ashkenazi,et al.  Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain. , 1993, The Journal of biological chemistry.

[21]  J. Nyborg,et al.  The GTP binding motif: variations on a theme , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[22]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[23]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[24]  B. Lee,et al.  The interpretation of protein structures: estimation of static accessibility. , 1971, Journal of molecular biology.

[25]  M. Sutcliffe,et al.  Definition of the switch surface in the solution structure of Cdc42Hs. , 1997, Biochemistry.

[26]  N. Nassar,et al.  Fluoride Activation of the Rho Family GTP-binding Protein Cdc42Hs* , 1998, The Journal of Biological Chemistry.

[27]  Katrin Rittinger,et al.  Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue , 1997, Nature.

[28]  S. Sprang,et al.  Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. , 1994, Science.

[29]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .