CRYSTAL STRUCTURE OF HUMAN RHOA IN A DOMINANTLY ACTIVE FORM COMPLEXED WITH A GTP ANALOGUE

K. Ihara, S. Muraguchi, M. Kato, T. Shimizu, M. Shirakawa, S. Kuroda, K. Kaibuchi and T. Hakoshima

Department of Bioscience, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan

Keywords: crystal structure, RhoA, small GTPase, dominantly active form, GTPgS

The 2.4-Å resolution crystal structure of a dominantly active form of the small GTPase RhoAV14(G14V mutant) complexed with the non-hydrolyzable GTP analogue, GTPgS reveals a fold similar to RhoA-GDP, which has been recently reported (1), but shows large conformational differences localized in switch I and II (2). These changes produce hydrophobic patches on the molecular surface of switch I, which suggested to be involved in its effector binding. Compared with H-Ras and other GTPases bound to GTP or GTP analogues, the significant conformational differences are located in regions involving switches I and II and part of antiparallel b- sheet between switches I and II (3). In addition to these differences, RhoA contains four insertion or deletion sites with an extra helical subdomain that seems to be characteristic of members of Rho family, including Rac1 (4) and Cdc42Hs (5), but with several variations in detail. These sites also display large displacement from those of H-Ras. The ADP-ribosylation residue, Asn41, by C3-like exoenzymes stacks on the indole ring of Trp58 with a hydrogen bond to the main chain of Glu40. The recognition of the guanosine moiety of GTPgS by RhoA contains water-mediated hydrogen bonds, which seems to be common in the Rho family GTPases. These structural differences provide an insight into specific interaction sites with the effectors, as well as with modulators such as GEF and GDI.

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