Nickel is an essential metal for a number of bacterial species that have developed systems for acquiring, delivering and incorporating the metal into target enzymes, and controlling the levels of nickel in cells to avoid toxic effects. As with other transition metals, these trafficking systems must be able to distinguish between the desired metal and other transition metal ions with similar physical and chemical properties. Because there are few enzymes (targets) that require nickel for activity (e.g., E. coli traffics nickel for hydrogenases made under anaerobic conditions and H. pylori requires nickel for hydrogenase and urease that are essential for acid viability), the ‘traffic pattern’ for nickel is relatively simple, and nickel trafficking therefore presents an opportunity to examine a system for the mechanisms that are used to distinguish nickel from other metals. In this review, we describe the details known for examples of uptake permeases, metallochaperones and proteins involved in metallocenter assembly, and nickel metalloregulators. We also illustrate the variety of mechanisms, including molecular recognition in the case of NikA protein and examples of allosteric regulation for HypA, NikR and RcnR, employed to generate specific biological responses to nickel ions.

Escherichia coli RcnR (resistance to cobalt and nickel regulator, EcRcnR), is a metal-responsive repressor of the genes encoding the Ni(II) and Co(II) exporter proteins RcnAB by binding to PrcnAB. DNA-binding affinity is weakened when the cognate ions Ni(II) or Co(II) bind to EcRcnR in a six-coordinate site that features a (N/O)5S ligand donor-atom set in distinct sites: while both metal ions are bound by the N-terminus, Cys35, and His64, Co(II) is additionally bound by His3. On the other hand, the non-cognate Zn(II) and Cu(I) ions feature a lower coordination number, have a solvent-accessible binding site, and coordinate protein ligands that do not include the N-terminal amine. A molecular model of apo-EcRcnR suggested potential roles for Glu34 and Glu63 in binding Ni(II) and Co(II) to EcRcnR. The roles of Glu34 and Glu63 in metal binding, metal selectivity and function were therefore investigated using a structure/function approach. X-ray absorption spectroscopy (XAS) was used to assess the structural changes in the Ni(II), Co(II), and Zn(II) binding sites of Glu → Ala and Glu → Cys variants at both positions. The effect of these structural alterations on the regulation of PrcnA by EcRcnR in response to metal binding was explored using LacZ reporter assays. These combined studies indicate that while Glu63 is a ligand for both metal ions, Glu34 is a ligand for Co(II) but possibly not for Ni(II). The Glu34 variants affect the structure of the cognate metal sites, but they have no effect on the transcriptional response. In contrast, the Glu63 variants affect both the structure and transcriptional response, although they do not completely abolish the function of EcRcnR. The structure of the Zn(II) site is not significantly perturbed by any of the Glu variations. The spectroscopic and functional data obtained on the mutants were used to calculate models of the metal site structures of EcRcnR bound to Ni(II), Co(II) and Zn(II). The results are interpreted in terms of a switch mechanism, in which a subset of the metal binding ligands is responsible for the allosteric response required for DNA release.

Intramolecular four-way junctions are structures present during homologous recombination, repair of double stranded DNA breaks, and integron recombination. Because of the wide range of biological processes four-way junctions are involved in, understanding how and under what conditions these structures form is critical. In this work, we used a combination of spectroscopic and calorimetric techniques to present a complete thermodynamic description of the unfolding of a DNA four-way junction (FWJ) and its appropriate control stem-loop motifs (Dumbbell, GAAATT-Hp, CTATC-Hp, GTGC-Hp, and GCGC-Hp). The overall results show that the four-way junction increases the cooperative unfolding of its stems, although the reason for this is unclear, as the arms do not unfold as coaxial stacks, and thus its melting behavior cannot be accurately described by its control molecules. This is in contrast to what has been seen for two- and three-way junctions. In addition, the lack of base stacking and the ΔH/ΔH ratio seen at low salt indicate the four-way junction exists as a mixture of conformations, one of which is most likely the open-X structure which has unpaired bases at the junction. This was confirmed by single value decomposition of CD and UV spectra. This indicates that at low salt there is a third spectroscopically distinct species, while at higher salt there are only two species, folded and unfolded. Based on the enthalpy, Δn, and Δn, the dominant folded structure at high salt is most likely the antiparallel stacked-X structure.

Oligonucleotide-directed triple helix formation has been recognized as a potential tool for targeting genes with high specificity. Cystosine methylation in the 5' position is both ubiquitous and a stable regulatory modification, which could potentially stabilize triple helix formation. In this work, we have used a combination of calorimetric and spectroscopic techniques to study the intramolecular unfolding of four triplexes and two duplexes. We used the following triplex control sequence, named Control Tri, d(AGAGACTCTCTCTCTCT), where C are loops of five cytosines. From this sequence, we studied three other sequences with dC → d(mC) substitutions on the Hoogsteen strand (2MeH), Crick strand (2MeC) and both strands (4MeHC). Calorimetric studies determined that methylation does increase the thermal and enthalpic stability, leading to an overall favorable free energy, and that this increased stability is cumulative, i.e. methylation on both the Hoogsteen and Crick strands yields the largest favorable free energy. The differential uptake of protons, counterions and water was determined. It was found that methylation increases cytosine protonation by shifting the apparent pK value to a higher pH; this increase in proton uptake coincides with a release of counterions during folding of the triplex, likely due to repulsion from the increased positive charge from the protonated cytosines. The immobilization of water was not affected for triplexes with methylated cytosines on their Hoogsteen or Crick strands, but was seen for the triplex where both strands are methylated. This may be due to the alignment in the major groove of the methyl groups on the cytosines with the methyl groups on the thymines which causes an increase in structural water along the spine of the triplex.

InrS (Internal nickel-responsive Sensor) is a transcriptional repressor of the nickel exporter NrsD and de-represses expression of the exporter upon binding Ni(II) ions. Although a crystal structure of apo-InrS has been reported, no structure of the protein with metal ions bound is available. Herein we report the results of metal site structural investigations of Ni(II) and Cu(II) complexes of InrS using x-ray absorption spectroscopy (XAS) that are complementary to data available from the apo-InrS crystal structure, and are consistent with a planar four-coordinate [Ni(His)2(Cys)2] structure, where the ligands are derived from the side chains of His21, Cys53, His78, and Cys82. Coordination of Cu(II) to InrS forms a nearly identical planar four-coordinate complex that is consistent with a simple replacement of the Ni(II) center by Cu(II).

Intramolecular three-way junctions are commonly found in both DNA and RNA. These structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication. In this work, we present a thermodynamic description of the unfolding of DNA intramolecular three-way junctions. We used a combination of spectroscopic and calorimetric techniques to investigate the folding/unfolding thermodynamics of two three-way junctions with a closed (Closed-J) or open (Open-J) junction and their appropriate control stem-loop motifs (GAAATT-Hp, CTATC-Hp, and Dumbbell). The overall results show that both junctions are stable over a wide range of salt concentrations. However, Open-J is more stable due to a higher enthalpy contribution from the formation of a higher number of basepair stacks whereas Closed-J has a defined structure and retains the basepair stacking of all three stems. The comparison of the experimental results of Closed-J and Open-J with those of their component stem-loop motifs allowed us to be more specific about their cooperative unfolding. For instance, Closed-J sacrifices thermal stability of the Dumbbell structure to maintain an overall folded state. At higher salt concentration, the simultaneous unfolding of the above domains is lost, resulting in the unfolding of the three separate stems. In contrast, the junction of Open-J in low salt retains the thermal and enthalpic stability of the Dumbbell structure although sacrificing stability of the CTATC stem. The relative stability of Dumbbell is the primary reason for the higher ΔG°, or free energy, value seen for Open-J at low salt. Higher salt not only maintains thermal stability of the Dumbbell structure in Open-J but causes the CTATC stem to fully fold.

Members of the uracil-DNA glycosylase (UDG) enzyme family recognize and bind uracil, sequestering it within the binding site pocket and catalyzing the cleavage of the base from the deoxyribose, leaving an abasic site. The recognition and binding are passive and rely on innate dynamic motions of DNA wherein base pairs undergo thermally induced breakage and conformational fluctuations. Once the uracil breaks from its base pair, it can be recognized and bound by the enzyme, which then alters its conformation for sequestration and catalysis. Our results suggest that the thymine to uracil substitution, which differs only by a single methyl group, causes a destabilization of the duplex thermodynamics, which would lead to an increase in the population of the extrahelical state and increase the probability of uracil being recognized and excised from DNA by UDG. This destabilization is dependent on the identity of the nearest-neighbor base-pair stacks; a G·C nearest neighbor leads to thermal and enthalpic destabilization that is weaker that that seen with two A·T neighbors. In addition, uracil substitution yields a nearest-neighbor increase in the counterion uptake of the duplexes but decreases the level of immobilization of structural water for all substituted duplexes regardless of the neighbor identity or number of substitutions.

We report the thermodynamic contributions of loop length and loop sequence to the overall stability of DNA intramolecular pyrimidine triplexes. Two sets of triplexes were designed: in the first set, the C loop closing the triplex stem was replaced with CTC loops (n = 1-5), whereas in the second set, both the duplex and triplex loops were replaced with a GCAA orAACG tetraloop. For the triplexes with a CTC loop, the triplex with five bases in the loop has the highest stability relative to the control. A loop length lower than five compromises the strength of the base-pair stacks without decreasing the thermal stability, leading to a decreased enthalpy, whereas an increase in the loop length leads to a decreased enthalpy and a higher entropic penalty. The incorporation of the GCAA loop yielded more stable triplexes, whereas the incorporation of AACG in the triplex loop yielded a less stable triplex due to an unfavorable enthalpy term. Thus, addition of the GCAA tetraloop can cause an increase in the thermodynamics of the triplex without affecting the sequence or melting behavior and may result in an additional layer of genetic regulation.