Structural and biochemical study of urease accessory protein complex provides mechanistic insights into the delivery of nickel to metalloenzyme urease, an enzyme enabling the survival of Helicobacter pylori in the human stomach.

SummaryFlagellar type III secretion systems (T3SS) contain an essential cytoplasmic‐ring (C‐ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non‐flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF‐T3SS C‐ring component in S higella flexneri is alternatively translated to produce both full‐length (Spa33‐FL) and a short variant (Spa33‐C), with both required for secretion. They associate in a 1:2 complex (Spa33‐FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33‐C 2 and identification of an unexpected intramolecular pseudodimer in Spa33‐FL reveal a molecular model for their higher order assembly within NF‐T3SS. Spa33‐FL and Spa33‐C are identified as functional counterparts of a FliM–FliN fusion and free FliN respectively. Furthermore, we show that T hermotoga maritima  FliM and FliN form a 1:3 complex structurally equivalent to Spa33‐FL/C2, allowing us to propose a unified model for C‐ring assembly by NF‐T3SS and flagellar‐T3SS.

The bacterial flagellum is a complex, self-assembling, nanomachine that confers motility to the cell. Despite great variation across species, all flagella are ultimately constructed from a helical propellor attached to a motor embedded in the inner membrane. The motor consists of a series of stator units surrounding a central rotor made up of two ring complexes, the MS-ring and the C-ring. Despite many studies, high resolution structural information is still completely lacking for the MS-ring of the rotor, and proposed mismatches in stoichiometry between the two rings have long provided a source of confusion for the field. We here present structures of the Salmonella MS-ring, revealing an unprecedented level of inter- and intra-chain symmetry variation that provides a structural explanation for the ability of the MS-ring to function as a complex and elegant interface between the two main functions of the flagellum, protein secretion and rotation.

Background: Maturation of urease is assisted by urease accessory proteins UreE, UreF, UreG, and UreH. Results: Crystal structure of UreF-UreH complex revealed conformational changes of UreF upon complex formation. Conclusion: Mutagenesis study confirmed that the conformational changes in UreF are essential for recruitment of UreG to the heterotrimeric complex of UreG-UreF-UreH. Significance: Our results provide a structural basis for understanding urease maturation.

The ability of metallochaperones to allosterically regulate the binding/release of metal ions and to switch protein-binding partners along the metal delivery pathway is essential to the metallation of the metalloenzymes. Urease, catalyzing the hydrolysis of urea into ammonia and carbon dioxide, contains two nickel ions bound by a carbamylated lysine in its active site. Delivery of nickel ions for urease maturation is dependent on GTP hydrolysis and is assisted by four urease accessory proteins UreE, UreF, UreG, and UreH(UreD). Here, we determined the crystal structure of the UreG dimer from in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound UreG (UreG) in the UreGFH complex reveals large conformational changes in the G2 region and residues near the CPH metal-binding motif. Upon GTP binding, the side chains of Cys66 and His68 from each of the UreG protomers rotate toward each other to coordinate a nickel ion in a square-planar geometry. Mutagenesis studies on UreG support the conformational changes induced by GTP binding as essential to dimerization of UreG, GTPase activity, in vitro urease activation, and the switching of UreG from the UreGFH complex to form the UreEG complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreGFH complex could activate urease in vitro in the presence of GTP. Based on our results, we propose a mechanism of how conformational changes of UreG during the GTP hydrolysis/binding cycle facilitate urease maturation.