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Intrinsic disorder and metal binding in UreG proteins from Archae hyperthermophiles: GTPase enzymes involved in the activation of Ni(II) dependent urease

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Abstract

Urease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein–protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii (Mj) and Metallosphaera sedula (Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG.

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Abbreviations

ITC:

Isothermal titration calorimetry

SEC:

Size-exclusion chromatography

MALS:

Multi-angle light scattering

QELS:

Quasi-elastic light scattering

RI:

Refraction index

CD:

Circular dichroism

TCEP:

Tris(2-carboxyethyl)phosphine

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Acknowledgments

Research supported by the University of Bologna and CIRMMP (Consorzio Interuniversitario di Risonanze Magnetiche di Metallo-Proteine). CERM (Center for Magnetic Resonance, University of Florence) is acknowledged for granting access to the NMR facility. Massimo Lucci and Fabio Calogiuri from CERM are acknowledged for data collection and useful discussions. We acknowledge Prof. Vladimir N. Uversky (USF, Tampa) for insightful discussions.

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  1. Manfredi Miraula

    Present address: Department of Chemistry, National University of Ireland, Maynooth, Ireland

Authors and Affiliations

  1. Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Viale Giuseppe Fanin 40, 40127, Bologna, Italy

    Manfredi Miraula, Stefano Ciurli & Barbara Zambelli

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  1. Manfredi Miraula
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  2. Stefano Ciurli
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Correspondence to Barbara Zambelli.

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Miraula, M., Ciurli, S. & Zambelli, B. Intrinsic disorder and metal binding in UreG proteins from Archae hyperthermophiles: GTPase enzymes involved in the activation of Ni(II) dependent urease. J Biol Inorg Chem 20, 739–755 (2015). https://doi.org/10.1007/s00775-015-1261-7

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