UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

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

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Abstract

Bacillus pasteurii UreG, a chaperons involved in the urease active site assembly, was overespressed 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 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 β-barrel surrounded by α-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.

Original languageEnglish (US)
Pages (from-to)4684-4695
Number of pages12
JournalJournal of Biological Chemistry
Volume280
Issue number6
DOIs
Publication statusPublished - Feb 11 2005

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Zambelli, B., Stola, M., Musiani, F., De Vriendt, K., Samyn, B., Devreese, B., ... Ciurli, S. (2005). UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+. Journal of Biological Chemistry, 280(6), 4684-4695. https://doi.org/10.1074/jbc.M408483200