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J. Biol. Chem., Vol. 279, Issue 45, 47352-47362, November 5, 2004

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Structural Determinants of RhoA Binding and Nucleotide Exchange in Leukemia-associated Rho Guanine-Nucleotide Exchange Factor^*

Romana Kristelly, Guang Gao, and John J. G. Tesmer¶

From the Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712-0165

Rho guanine-nucleotide exchange factors (RhoGEFs) activate Rho GTPases, and thereby regulate cytoskeletal structure, gene transcription, and cell migration. Leukemia-associated RhoGEF (LARG) belongs to a small subfamily of RhoGEFs that are RhoA-selective and directly activated by the G_12/13 family of heterotrimeric G proteins. Herein we describe the atomic structures of the catalytic Dbl homology (DH) and pleckstrin homology (PH) domains of LARG alone and in complex with RhoA. These structures demonstrate that the DH/PH domains of LARG can undergo a dramatic conformational change upon binding RhoA, wherein both the DH and PH domains directly engage RhoA. Through mutational analysis we show that full nucleotide exchange activity requires a novel N-terminal extension on the DH domain that is predicted to exist in a broader family of RhoGEFs that includes p115-RhoGEF, Lbc, Lfc, Net1, and Xpln, and identify regions within the LARG PH domain that contribute to its ability to facilitate nucleotide exchange in vitro. In crystals of the DH/PH-RhoA complex, the active site of RhoA adopts two distinct GDP-excluding conformations among the four unique complexes in the asymmetric unit. Similar changes were previously observed in structures of nucleotide-free Ras and Ef-Tu. A potential protein-docking site on the LARG PH domain is also evident and appears to be conserved throughout the Lbc subfamily of RhoGEFs.

Received for publication, June 1, 2004 , and in revised form, August 18, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) 1TXD and 1X86.

^* This work was supported by Welch Foundation chemical research Grant F-1487, American Heart Association Scientist Development Grant 0235273N, Research Corporation Cottrell Scholar award CS0940, and by the College of Natural Sciences support to the Center for Structural Biology (University of Texas at Austin). Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract W-31-109-Eng-38. Use of the BioCARS Sector 14 was supported by the National Institutes of Health, National Center for Research Resources, under Grant RR07707. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station #A5300, Austin, TX 78712-0165. Tel.: 512-471-5675; Fax: 512-471-8696; E-mail: tesmer{at}mail.utexas.edu.

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