Structural insights into how GTP-dependent conformational changes in a metallochaperone UreG facilitate urease maturation

Abstract

<p id="d10065184e200">Our work provides insights into how cells solve the problem of delivering nickel, a toxic metal, to the active site of a metalloenzyme such as urease. Urease, a nickel-containing enzyme, is a virulence factor for <i>Helicobacter pylori</i>, which infects half of the human population and causes peptic ulcers. Supported by structural and biochemical evidence, we present a paradigm on how a metallochaperone UreG couples GTP hydrolysis/binding to allosterically control the binding/release of nickel ions and to switch protein-binding partners along the metal-delivery pathway so that the nickel ions are passing from one metallochaperone to another, without releasing the “free” toxic metal to the cytoplasm. </p><p class="first" id="d10065184e206">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 <i>Klebsiella pneumoniae</i> in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound <i>Helicobacter pylori</i> UreG ( <i>Hp</i>UreG) in the UreG ₂F ₂H ₂ complex reveals large conformational changes in the G2 region and residues near the ₆₆CPH ₆₈ 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 <i>Hp</i>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 UreG ₂F ₂H ₂ complex to form the UreE ₂G ₂ complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreG ₂F ₂H ₂ 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. </p>