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1 Protein Engineering Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA
2 Structural Biology Core Facility, Center For Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA
Reprint requests to: David S. Waugh, Protein Engineering Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702-1201, USA; e-mail: waughd{at}ncifcrf.gov; fax: (301) 846-7148.
Yersinia pestis, the causative agent of bubonic plague, evades the immune response of the infected organism by using a type III (contact-dependent) secretion system to deliver effector proteins into the cytosol of mammalian cells, where they interfere with signaling pathways that regulate inflammation and cytoskeleton dynamics. The cytotoxic effector YopE functions as a potent GTPase-activating protein (GAP) for Rho family GTP-binding proteins, including RhoA, Rac1, and Cdc42. Down-regulation of these molecular switches results in the loss of cell motility and inhibition of phagocytosis, enabling Y. pestis to thrive on the surface of macrophages. We have determined the crystal structure of the GAP domain of YopE (YopEGAP; residues 90–219) at 2.2-Å resolution. Apart from the fact that it is composed almost entirely of 
-helices, YopEGAP shows no obvious structural similarity with eukaryotic RhoGAP domains. Moreover, unlike the catalytically equivalent arginine fingers of the eukaryotic GAPs, which are invariably contained within flexible loops, the critical arginine in YopEGAP (Arg144) is part of an -helix. The structure of YopEGAP is strikingly similar to the GAP domains from Pseudomonas aeruginosa (ExoSGAP) and Salmonella enterica (SptPGAP), despite the fact that the three amino acid sequences are not highly conserved. A comparison of the YopEGAP structure with those of the Rac1-ExoSGAP and Rac1-SptP complexes indicates that few, if any, significant conformational changes occur in YopEGAP when it interacts with its G protein targets. The structure of YopEGAP may provide an avenue for the development of novel therapeutic agents to combat plague.
Keywords: GAP; GTPase-activating protein; plague; cytotoxin; cytoskeleton; Rho
Abbreviations: GAP, GTPase activating protein • TEV, tobacco etch virus • IPTG, isopropyl-ß-d-thiogalactopyranoside • DTT, dithiothreitol • EDTA, ethylenediaminetetraacetic acid • CCD, charge-coupled device
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