The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

nal transduction pathways that regulate actin cytoskele-ton dynamics and inflammation (Cornelis, 2002). The deployment of cytotoxic effectors is regulated in part by the action of the LcrV protein (V-antigen), an essential 2 Macromolecular Crystallography Laboratory National Cancer Institute at Frederick virulence factor with multiple functions. In the bacterial cytoplasm, LcrV stimulates type III secretion by binding Frederick, Maryland 21702 3 Procter & Gamble Pharmaceuticals to the negative regulatory protein LcrG and neutralizing its ability to block secretion (Nilles et al., 1997). LcrV is Health Care Research Center-Discovery 8700 Mason-Montgomery Road also exposed at the bacterial cell surface prior to contact with mammalian cells (Fields et al., 1999; Pettersson et Mason, Ohio 45040 al., 1999), and may play a role in cell-cell adhesion. Some evidence suggests that LcrV can form pores in eukaryotic cell membranes (Holmströ m et al., 2001), Summary and, in conjunction with the Yersinia YopB and YopD proteins, it probably forms the translocation pore through The LcrV protein (V-antigen) is a multifunctional viru-which the effectors are delivered into mammalian cells. lence factor in Yersinia pestis, the causative agent of Additionally, LcrV is both secreted into the extracellular plague. LcrV regulates the translocation of cytotoxic milieu and translocated into eukaryotic cells (Fields and effector proteins from the bacterium into the cytosol Straley, 1999), where it exerts strong immunomodula-of mammalian cells via a type III secretion system, tory effects (Nakajima et al., 1995; Nedialkov et al., 1997; possesses antihost activities of its own, and is also an Welkos et al., 1998). active and passive mediator of resistance to disease. The complex array of functions attributed to LcrV has Although a crystal structure of this protein has been effectively stymied efforts to elucidate its mechanisms actively sought for better understanding of its role of action. Attempts to study structure-function relation-in pathogenesis, the wild-type LcrV was found to be ships by deletion analysis and site-directed mutagene-recalcitrant to crystallization. We employed a surface sis have yielded little useful information because they entropy reduction mutagenesis strategy to obtain crys-have been carried out without any knowledge about the tals of LcrV that diffract to 2.2 A ˚ and determined its tertiary structure of LcrV. With that in mind, we set out structure. The refined model reveals a dumbbell-like to determine the crystal structure of LcrV. molecule with a novel fold that includes an unexpected coiled-coil motif, and provides a detailed three-dimensional roadmap for exploring structure-function rela-Results and Discussion tionships in this essential virulence determinant. Structure Determination Because crystals of the wild-type LcrV protein could not Introduction be obtained, we resorted to a novel strategy based on mutational modification of surface properties to reduce The three main plague pandemics are thought to...

Assembly of the bacterial flagellum and type III secretion in pathogenic bacteria require cytosolic export chaperones that interact with mobile components to facilitate their secretion. Although their amino acid sequences are not conserved, the structures of several type III secretion chaperones revealed striking similarities between their folds and modes of substrate recognition. Here, we report the first crystallographic structure of a flagellar export chaperone, Aquifex aeolicus FliS. FliS adopts a novel fold that is clearly distinct from those of the type III secretion chaperones, indicating that they do not share a common evolutionary origin. However, the structure of FliS in complex with a fragment of FliC (flagellin) reveals that, like the type III secretion chaperones, flagellar export chaperones bind their target proteins in extended conformation and suggests that this mode of recognition may be widely used in bacteria.