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J. Biol. Chem., Vol. 280, Issue 6, 4684-4695, February 11, 2005

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UreG, a Chaperone in the Urease Assembly Process, Is an Intrinsically Unstructured GTPase That Specifically Binds Zn^2+*

Barbara Zambelli, Massimiliano Stola¶, Francesco Musiani, Kris De Vriendt||, Bart Samyn||, Bart Devreese||**, Jozef Van Beeumen||**, Paola Turano, Alexander Dikiy¶, Donald A. Bryant, and Stefano Ciurli¶¶

From the Laboratory of Bioinorganic Chemistry, Department of Agro-Environmental Science and Technology, University of Bologna, I-40127 Bologna, Italy, ^||Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology, and Microbiology, University of Gent, B-9000 Gent, Belgium, Magnetic Resonance Center and Department of Chemistry, University of Firenze, I-50019 Firenze, Italy, and the Department of Biochemistry and Molecular Biology, the Pennsylvania State University, University Park, Pennsylvania 16802

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 k_cat = 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 K_D = 42 ± 3 µM, 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 -barrel surrounded by -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.

Received for publication, July 27, 2004 , and in revised form, November 9, 2004.

^* The work was supported in part by Grants PRIN-2001 and PRIN-2003 from the Ministero Italiano dell'Università e della Ricerca. Work in the laboratory of D. A. B. was supported by National Institutes of Health Grant GM-31625. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Ph.D. fellowship provided by the University of Bologna.

^¶ Recipient of fellowship provided by Consorzio Interuniversitario per le Risonanze Magnetiche di Metalloproteine Paramagnetiche.

^** Supported by the Funds for Scientific Research-Flanders Grant G.0190.04.

^¶¶ To whom correspondence should be addressed: Laboratory of Bioinorganic Chemistry, Dept. of Agro-Environmental Science and Technology, University of Bologna, Viale Giuseppe Fanin 40, 40127 Bologna, Italy. Tel.: 39-051-209-6204; Fax: 39-051-209-6203; E-mail: stefano.ciurli{at}unibo.it.

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