Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of ~120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix α 1 -α 2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two helix bundle, and only residues from helix α 1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.

The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, x-ray crystallography and hydrogen-deuterium mass spectrometry we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure.

The hantaviruses are emerging infectious viruses that in humans can cause a cardiopulmonary syndrome or a hemorrhagic fever with renal syndrome. The nucleocapsid (N) is the most abundant viral protein, and during viral assembly, the N protein forms trimers and packages the viral RNA genome. Here, we report the NMR structure of the N-terminal domain (residues 1-74, called N 1-74 ) of the Andes hantavirus N protein. N 1-74 forms two long helices (␣ 1 and ␣ 2 ) that intertwine into a coiled coil domain. The conserved hydrophobic residues at the helix ␣ 1 -␣ 2 interface stabilize the coiled coil; however, there are many conserved surface residues whose function is not known. Site-directed mutagenesis, CD spectroscopy, and immunocytochemistry reveal that a point mutation in the conserved basic surface formed by Arg 22 or Lys 26 lead to antibody recognition based on the subcellular localization of the N protein. Thus, Arg 22 and Lys 26 are likely involved in a conformational change or molecular recognition when the N protein is trafficked from the cytoplasm to the Golgi, the site of viral assembly and maturation.Hantaviruses can cause two emerging infectious diseases known as the hantavirus cardiopulmonary syndrome (HCPS) 3 and the hantavirus hemorrhagic fever with renal syndrome (1). Annually, there are over 150,000 cases of hantaviral infections reported world wide (2). Rodents are the primary reservoir of hantaviruses, and humans are normally infected by inhalation of aerosol contaminated with the excreta of infected rodents. The first reported cases of HCPS in North America (3) was caused by a novel hantaviral species (4, 5), the Sin Nombre virus, and had an initial mortality rate of 78%. HCPS has since been reported throughout the United States with a current mortality rate of 35% when correctly diagnosed (6). The major cause of HCPS in South America is the Andes virus, and person-to-person transmission of the Andes virus was reported in Argentina and Chile (7). Hantaviruses are known to invade and replicate primarily in endothelial cells, including the endothelium of vascular tissues lining the heart (8 -10).The genome of hantaviruses consists of three negativestranded RNAs, which encode the nucleocapsid (N) protein, two integral membrane glycoproteins (G1 and G2), and an RNAdependent RNA polymerase (L protein). The N protein is highly immunogenic (11, 12) and elicits a strong immune response, which confers protection in mice (13-15). It is highly conserved and is the most abundant viral protein, and it plays important roles in viral encapsidation, RNA packaging, and host-pathogen interaction (16). The N protein binds to viral proteins (16), host proteins (17-23), and viral RNA (24 -28). The self-association of the N protein into trimers was shown by gradient fractionation and chemical cross-linking (29). Deletion mapping identified that regions at the N and C termini are important in N-N interaction (29 -31), and a model of trimerization was proposed based on the head-to-head and tail-to-tail association of the...

No abstract