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The uptake of essential metal ions and the ability to extrude them when their excess causes toxicity are crucial processes for all living beings. Nickel is a virulence factor for several human pathogens and in particular for the human gastric pathogen Helicobacter pylori because of its crucial role in the catalytic activity of two Ni-dependent enzymes, urease and hydrogenase. H. pylori requires efficient uptake mechanisms to import Ni(II) because of its scarcity in the human body, but the molecular details of Ni(II) homeostasis are not fully known. Here we offer a structural framework for the machinery of Ni(II) import/export in H. pylori, obtained through comparative modelling and macromolecular docking. The model structures reported in this perspective are initial steps towards the understanding of these processes at the molecular level and in the direction to exploit them to eradicate infections caused by this family of pathogens. The differences between the structural models obtained by using both the recently released neural network-based approach implemented in AlphaFold2 and a more classical user-driven modelling procedure are also discussed., (© The Author(s) 2021. Published by Oxford University Press.)

The survival of several pathogenic bacteria, such as Helicobacter pylori (Hp), relies on the activity of the nickel-dependent enzyme urease. Nickel insertion into urease is mediated by a multimeric chaperone complex (HpUreDFG) that is responsible for the transport of Ni(II) from a conserved metal binding motif located in the UreG dimer (CPH motif) to the catalytic site of the enzyme. The X-ray structure of HpUreDFG revealed the presence of water-filled tunnels that were proposed as a route for Ni(II) translocation. Here, we probe the transport of Ni(II) through the internal tunnels of HpUreDFG, from the CPH motif to the external surface of the complex, using microsecond-long enhanced molecular dynamics simulations. The results suggest a "bucket-brigade" mechanism whereby Ni(II) can be transported through a series of stations found along these internal pathways., (Copyright © 2021 Elsevier Inc. All rights reserved.)

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter ( P hp1227 ). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the P hp1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

Urease is a nickel-containing enzyme that is essential for the survival of several and often deadly pathogenic bacterial strains, including Helicobacter pylori . Notwithstanding several attempts, the development of direct urease inhibitors without side effects for the human host remains, to date, elusive. The recently solved X-ray structure of the Hp UreDFG accessory complex involved in the activation of urease opens new perspectives for structure-based drug discovery. In particular, the quaternary assembly and the presence of internal tunnels for nickel translocation offer an intriguing possibility to target the Hp UreDFG complex in the search of indirect urease inhibitors. In this work, we adopted a theoretical framework to investigate such a hypothesis. Specifically, we searched for putative binding sites located at the protein-protein interfaces on the Hp UreDFG complex, and we challenged their druggability through structure-based virtual screening. We show that, by virtue of the presence of tunnels, some protein-protein interfaces on the Hp UreDFG complex are intrinsically well suited for hosting small molecules, and, as such, they possess good potential for future drug design endeavors.

Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using 1 H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.

Transition metals are both essential micronutrients and limited in environmental availability. The Ni(II)-dependent urease protein, the most efficient enzyme known to date, is a paradigm for studying the strategies that cells use to handle an essential, yet toxic, metal ion. Urease is a virulence factor of several human pathogens, in addition to decreasing the efficiency of soil organic nitrogen fertilization. Ni(II) insertion in the urease active site is performed through the action of three essential accessory proteins: UreD, UreF, and UreG. The crystal structure of the UreD-UreF-UreG complex from the human pathogen Helicobacter pylori (HpUreDFG) revealed the presence of tunnels that cross the entire length of both UreF and UreD, potentially able to deliver Ni(II) ions from UreG to apo-urease. Atomistic molecular dynamics simulations performed on the HpUreDFG complex in explicit solvent and at physiological ionic conditions demonstrate the stability of these protein tunnels in solution and provide insights on the trafficking of water molecules inside the tunnels. The presence of different alternative routes across the identified tunnels for Ni(II) ions, water molecules, and carbonate ions, all involved in urease activation, is highlighted here, and their potential role in the urease activation mechanism is discussed.

The interaction between macromolecules is a fundamental aspect of most biological processes. The computational techniques used to study protein-protein and protein-nucleic acid interactions have evolved in the last few years because of the development of new algorithms that allow the a priori incorporation, in the docking process, of experimentally derived information, together with the possibility of accounting for the flexibility of the interacting molecules. Here we review the results and the evolution of the techniques used to study the interaction between metallo-proteins and DNA operators, all involved in the nickel and iron metabolism of pathogenic bacteria, focusing in particular on Helicobacter pylori (Hp). In the first part of the article we discuss the methods used to calculate the structure of complexes of proteins involved in the activation of the nickel-dependent enzyme urease. In the second part of the article, we concentrate on two applications of protein-DNA docking conducted on the transcription factors HpFur (ferric uptake regulator) and HpNikR (nickel regulator). In both cases we discuss the technical expedients used to take into account the conformational variability of the multi-domain proteins involved in the calculations.

Helicobacter pylori UreF (HpUreF) is involved in the insertion of Ni(2+) 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 Ni(2+) ions per dimer, with a micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopy indicated that the Ni(2+) ions reside in a five-coordinate pyramidal geometry comprising exclusively N/O-donor ligands derived from the protein, including one or two histidine imidazole and carboxylate ligands. Binding of Ni(2+) 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 interact with H. pylori UreD, altered the affinity of the protein for Ni(2+). This result, complemented by the findings from X-ray absorption spectroscopy, indicates that the Ni(2+) 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 Ni(2+) 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 apoenzyme-to-holoenzyme activation.

Most transcriptional regulators bind nucleotide motifs in the major groove, although some are able to recognize molecular determinants conferred by the minor groove of DNA. Here we report a transcriptional commutator switch that exploits the alternative readout of grooves to mediate opposite output regulation for the same input signal. This mechanism accounts for the ability of the Helicobacter pylori Fur regulator to repress the expression of both iron-inducible and iron-repressible genes. When iron is scarce, Fur binds to DNA as a dimer, through the readout of thymine pairs in the major groove, repressing iron-inducible transcription (FeON). Conversely, on iron-repressible elements the metal ion acts as corepressor, inducing Fur multimerization with consequent minor groove readout of AT-rich inverted repeats (FeOFF). Our results provide first evidence for a novel regulatory paradigm, in which the discriminative readout of DNA grooves enables to toggle between the repression of genes in a mutually exclusive manner.

The molecular details of the protein complex formed by UreD, UreF, UreG, and UreE, accessory proteins for urease activation in the carcinogenic bacterium Helicobacter pylori, have been elucidated using computational modeling. The calculated structure of the complex supports the hypothesis of UreF acting as a GTPase activation protein that facilitates GTP hydrolysis by UreG during urease maturation, and provides a rationale for the design of new drugs against infections by ureolytic bacterial pathogens.

The pathogenicity of Helicobacter pylori depends on the activity of urease for pH modification. Urease activity requires assembly of a dinickel active site that is facilitated in part by GTP hydrolysis by UreG. The proper functioning of Helicobacter pylori UreG (HpUreG) is dependent on Zn(II) binding and dimerization. X-ray absorption spectroscopy and structural modeling were used to elucidate the structure of the Zn(II) site in HpUreG. These studies independently indicated a site at the dimer interface that has trigonal bipyramidal geometry and is composed of two axial cysteines at 2.29(2) Å, two equatorial histidines at 1.99(1) Å, and a solvent-accessible coordination site. The final model for the Zn(II) site structure was determined by refining multiple-scattering extended X-ray absorption fine structure fits using the geometry predicted by homology modeling and ab initio calculations.

The analysis of the sequence of Helicobacter pylori UreD(H), an accessory protein involved in the activation of urease through the assembly of the Ni(2+)-containing active site, revealed the presence of two domains. The structure of these domains was calculated using threading and modeling algorithms. A search for putative binding sites on the protein surface was carried out using dedicated algorithms sensitive to either sequence conservation or structural similarity based on geometry and physicochemical properties. The results suggest that UreD(H) acts as a multifunctional molecular recognition platform facilitating the interaction between apo-urease and the ancillary proteins UreG, UreF, and UreE, responsible for nickel trafficking and delivering.

The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni(2+) for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni(2+) insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni(2+) (Kd=0.15 microM) or Zn(2+) (Kd=0.49 microM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn(2+) and Ni(2+), which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE-HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn(2+), and not for Ni(2+), in the stabilization of this complex was demonstrated using size-exclusion chromatography, light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of a novel binding site for Zn(2+), actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn(2+) in the Ni(2+)-dependent urease system is envisaged.

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism., ((c) 2008 Wiley-Liss, Inc.)

Helicobacter pylori ( Hp) is a carcinogen that relies on Ni(II) to survive in the extreme pH conditions of the human guts. The regulation of genes coding for Ni(II) enzymes and proteins is effected by the nickel-responsive transcription factor NikR, composed of a DNA-binding domain (DBD) and a metal-binding domain (MBD). The scope of this study is to obtain the molecular details of the HpNikR interaction with the urease operator OP, in solution. The size of the full-length protein prevents the characterization of the HpNikR-OP interaction using NMR. We thus investigated the two separate domains of HpNikR. The conservation of their oligomeric state was established by multiple-angle light scattering. Isothermal calorimetric titrations indicated that the thermodynamics of Ni(II) binding to the isolated MBD is independent of the presence of the adjacent DBDs. The NMR spectra of the isolated DBD support considerable conservation of its structural properties. The spectral perturbations induced on the DBD by OP provided information useful to calculate a structural model of the HpNikR-OP complex using a docking computational protocol. The NMR assignment of the residues involved in the protein-DNA interaction represents a starting point for the development of drugs potentially able to eradicate H. pylori infections. All evidences so far collected, in this and previous studies, consistently indicate that binding of Ni(II) to the MBD increases the HpNikR-DNA affinity by modulating the dynamic, and not the structural, properties of the protein, suggesting that the formation of a stable complex relies upon an induced fit mechanism. [ABSTRACT FROM AUTHOR]

UreG proteins are small GTP binding (G) proteins that catalyze the hydrolysis of GTP necessary for the maturation of urease, a virulence factor in bacterial pathogenesis. UreG proteins are the first documented cases of intrinsically disordered enzymes. The comprehension of the dynamics of folding–unfolding events occurring in this protein could shed light on the enzymatic mechanism of UreG. Here, we used the recently developed replica exchange with solute tempering (REST2) computational methodology to explore the conformational space of UreG from Helicobacter pylori (HpUreG) and to identify its structural fluctuations. The same simulation and analysis protocol has been applied to HypB from Methanocaldococcus jannaschii (MjHypB), which is closely related to UreG in both sequence and function, even though it is not intrinsically disordered. A comparison of the two systems reveals that both HpUreG and MjHypB feature a substantial rigidity of the protein regions involved in catalysis, justifying its residual catalytic activity. On the other hand, HpUreG tends to unfold more than MjHypB in portions involved in protein-protein interactions with metallochaperones necessary for the formation of multiprotein complexes known to be involved in urease activation. [ABSTRACT FROM AUTHOR]