Model Structures of Helicobacter pylori UreD(H) Domains: A Putative Molecular Recognition Platform

Musiani, Francesco; Bellucci, Matteo; Ciurli, Stefano

doi:10.1021/ci200183n

Cited by 14 publications

(7 citation statements)

References 64 publications

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“…A similar role could also involve UreD. 42 In this view, native disorder is a possible general mechanism that cells use to regulate enzymatic activity, allowing UreG, partially or totally inactive in the isolated state, to interact and to be regulated by different protein partners.…”

Section: Discussionmentioning

confidence: 99%

Insights in the (un)structural organization of Bacillus pasteurii UreG, an intrinsically disordered GTPase enzyme

et al. 2012

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In the past, enzymatic activity has always been expected to be dependent on overall protein rigidity, necessary for substrate recognition and optimal orientation. However, increasing evidence is now accumulating, revealing that some proteins characterized by intrinsic disorder are actually able to perform catalysis. Among them, the only known natural intrinsically disordered enzyme is UreG, a GTPase that, in plants and bacteria, is involved in the protein interaction network leading to Ni(2+) ions delivery into the active site of urease. In this paper, we report a detailed analysis of the unfolding behaviour of UreG from Bacillus pasteurii (BpUreG), following its thermal and chemical denaturation with a combination of fluorescence spectroscopy, calorimetry, CD and NMR. The results demonstrate that BpUreG exists as an ensemble of inter-converting conformations, whose degrees of secondary structure depend on temperature and denaturant concentration. In particular, three major types of conformational ensembles with different degrees of residual structure were identified, with major structural characteristics resembling those of a molten globule (low temperature, absence of denaturant), pre-molten globule (high temperature, absence or presence of denaturant) and random coil (low temperature, presence of denaturant). Transitions among these ensembles of conformational states occur non-cooperatively although reversibly, with a gradual loss or acquisition of residual structure depending on the conditions. A possible role of disorder in the biological function of UreG is envisaged and discussed.

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Section: Discussionmentioning

confidence: 99%

Insights in the (un)structural organization of Bacillus pasteurii UreG, an intrinsically disordered GTPase enzyme

et al. 2012

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“…Overall, the shift in population resulting from peptide binding could be one of the keys to facilitate subsequent binding of additional partners at yet unidentified sites of NarJ. Such a situation may also hold true in other metalloproteins of interest such as hydrogenases or ureases which rely on dedicated chaperones for their folding and function [73], [74].…”

Section: Discussionmentioning

confidence: 99%

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

et al. 2012

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Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.

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“…UreD is the first protein to come into direct contact with urease, as revealed by chemical cross-linking experiments and mass spectrometry for Ka UreD [21] as well as two-hybrid analyses and immuno-precipitation studies for Proteus mirabilis ( Pm ) UreD [22]. UreD mediates the interaction with other chaperones, likely acting as a protein platform [23, 24]. UreF binds the apo urease-UreD complex by directly interacting with UreD, as shown using chemical crosslinking and small-angle X-ray scattering (SAXS) experiments on K. aerogenes proteins [21, 25], and two-hybrid studies on P. mirabilis [22] and H. pylori [26] proteins.…”

Section: Introductionmentioning

confidence: 99%

Nickel binding properties of Helicobacter pylori UreF, an accessory protein in the nickel-based activation of urease

et al. 2013

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Helicobacter pylori UreF is involved in the insertion of Ni2+ in the urease active site. The recombinant protein in solution is a dimer characterized by an extensive α-helical structure and a well-folded tertiary structure. HpUreF binds two Ni2+ ions per dimer, with micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopic analysis indicates that the Ni2+ ions reside in a five-coordinate pyramidal geometry comprised exclusively of N/O-donor ligands derived from the protein, including one or two histidine imidazoles and carboxylate ligands. Binding of Ni2+ does not affect the solution properties of the protein. Mutation to alanine of His229 and/or Cys231, a pair of residues located on the protein surface that interacts with HpUreD, altered the affinity of the protein for Ni2+. This result, complemented by the XAS analysis, indicates that the Ni2+ binding site involves His229, and that Cys231 has an indirect structural role in metal binding. An in vivo assay of urease activation demonstrated that H229A-HpUreF, C231A-HpUreF and H229/C231-HpUreF are significantly less competent in this process, suggesting a role for a Ni2+ complex with UreF in urease maturation. This hypothesis was supported by calculations revealing the presence of a tunnel that joins the Cys-Pro-His metal binding site on UreG and an opening on the UreD surface, passing through UreF close to His229 and Cys231, in the structure of the H. pylori UreDFG complex. This tunnel could be used to transfer nickel into the urease active site during apo-to-holo enzyme activation.

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Model Structures of Helicobacter pylori UreD(H) Domains: A Putative Molecular Recognition Platform

Cited by 14 publications

References 64 publications

Insights in the (un)structural organization of Bacillus pasteurii UreG, an intrinsically disordered GTPase enzyme

Insights in the (un)structural organization of Bacillus pasteurii UreG, an intrinsically disordered GTPase enzyme

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

Nickel binding properties of Helicobacter pylori UreF, an accessory protein in the nickel-based activation of urease

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