RESUMEN
Urease is a nickel (Ni) enzyme that is essential for the colonization of Helicobacter pylori in the human stomach. To solve the problem of delivering the toxic Ni ion to the active site without diffusing into the cytoplasm, cells have evolved metal carrier proteins, or metallochaperones, to deliver the toxic ions to specific protein complexes. Ni delivery requires urease to form an activation complex with the urease accessory proteins UreFD and UreG. Here, we determined the cryo-electron microscopy structures of H. pylori UreFD/urease and Klebsiella pneumoniae UreD/urease complexes at 2.3- and 2.7-angstrom resolutions, respectively. Combining structural, mutagenesis, and biochemical studies, we show that the formation of the activation complex opens a 100-angstrom-long tunnel, where the Ni ion is delivered through UreFD to the active site of urease.
Asunto(s)
Helicobacter pylori , Ureasa , Humanos , Ureasa/química , Ureasa/metabolismo , Dominio Catalítico , Microscopía por Crioelectrón , Proteínas Bacterianas/metabolismo , Helicobacter pylori/química , Níquel/química , Níquel/metabolismo , KlebsiellaRESUMEN
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 Klebsiella pneumoniae in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound Helicobacter pylori UreG (HpUreG) in the UreG2F2H2 complex reveals large conformational changes in the G2 region and residues near the 66CPH68 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 HpUreG 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 UreG2F2H2 complex to form the UreE2G2 complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreG2F2H2 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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Guanosina Trifosfato/metabolismo , Metalochaperonas/química , Metalochaperonas/metabolismo , Conformación Proteica , Ureasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Activación Enzimática , Guanosina Trifosfato/química , Metalochaperonas/genética , Modelos Biológicos , Modelos Moleculares , Mutación , Níquel/química , Níquel/metabolismo , Proteínas de Unión a Fosfato , Unión Proteica , Multimerización de Proteína , Relación Estructura-ActividadRESUMEN
Homology modeling allows the prediction of a protein structure based on sequence similarity to a known structure of homologous proteins. In this chapter, we use a plant-specific AtSar1a-Atsec23a pair of proteins as a case study to illustrate how to use homology modeling to understand the specificity of the pairwise interaction between AtSar1a and AtSec23a. The detailed procedures described here are also useful in structure prediction of other protein complexes.
