title: UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+.
abstract: Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.
authors.affiliation: Laboratory of Bioinorganic Chemistry, Department of Agro-Environmental Science and Technology, University of Bologna, I-40127 Bologna, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Zambelli
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Stola
authors.forename: Francesco
authors.initials: F
authors.lastname: Musiani
authors.forename: Kris
authors.initials: K
authors.lastname: De Vriendt
authors.forename: Bart
authors.initials: B
authors.lastname: Samyn
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Paola
authors.initials: P
authors.lastname: Turano
authors.forename: Alexander
authors.initials: A
authors.lastname: Dikiy
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bryant
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D007834:Lasers; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008670:Metals / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D028044:Phosphate-Binding Proteins; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D014510:Urease / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D007477:Ions; D008670:Metals; D028044:Phosphate-Binding Proteins; D011994:Recombinant Proteins; C082270:ureG protein, Bacteria; D009532:Nickel; D014510:Urease; D020558:GTP Phosphohydrolases; D015032:Zinc