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Background: Injuries are a common occurrence in military recruit training, however due to differences in the capture of training exposure, injury incidence rates are rarely reported. Our aim was to determine the musculoskeletal injury epidemiology of military recruits, including a standardised injury incidence rate. Methods: Epidemiological systematic review following the PRISMA 2020 guidelines. Five online databases were searched from database inception to 5th May 2021. Prospective and retrospective studies that reported data on musculoskeletal injuries sustained by military recruits after the year 2000 were included. We reported on the frequency, prevalence and injury incidence rate. Incidence rate per 1000 training days (Exact 95% CI) was calculated using meta-analysis to allow comparisons between studies. Observed heterogeneity (e.g., training duration) precluded pooling of results across countries. The Joanna Briggs Institute Quality Assessment Checklist for Prevalence Studies assessed study quality. Results: This review identified 41 studies comprising 451,782 recruits. Most studies (n=26; 63%) reported the number of injured recruits, and the majority of studies (n=27; 66%) reported the number of injuries to recruits. The prevalence of recruits with medical attention injuries or time-loss injuries was 22.8% and 31.4%, respectively. Meta-analysis revealed the injury incidence rate for recruits with a medical attention injury may be as high as 19.52 injuries per 1000 training days; and time-loss injury may be as high as 3.97 injuries per 1000 training days. Longer recruit training programs were associated with a reduced injury incidence rate (p=0.003). The overall certainty of the evidence was low per a modified GRADE approach. Conclusion: This systematic review with meta-analysis highlights a high musculoskeletal injury prevalence and injury incidence rate within military recruits undergoing basic training with minimal improvement observed over the past 20 years. Longer training program, which may decrease the degree of overload experienced by recruit, may reduce injury incidence rates. Unfortunately, reporting standards and reporting consistency remain a barrier to generalisability. Systematic Review Registration: PROSPERO (Registration number: CRD42021251080)

To determine the concurrent validity of player self-reported and independently observed throwing volume. Examine whether sex, playing position, or time to upload self-reported data post training influences the accuracy of self-reported throwing loads.Cross-sectional cohort study.A total of 8 female and 18 male elite cricket players participated in the study. Overarm throws from 12 training sessions during the 2020-21 cricket year were observed. Player self-reported throwing volume data were retrieved post training, with the time difference between session completion and self-reported data upload recorded.A moderate positive correlation was found between self-reported and observed throwing loads (rho = 0.65), however only 22 % of players reported values within a 10 % level of error. Players reported a mean (SD) absolute inaccuracy of 11.17 (9.77) throws, and a mean (SD) relative inaccuracy of 24.76 (16.04) percent. Sex did not influence reporting accuracy (p = 0.41). Females tended to upload self-reported data the day of training, whereas men report the day following. Players who uploaded their data greater than one day after training were the most inaccurate with a mean relative inaccuracy of 36 %.While there is a clear relationship between observed and self-reported throwing volumes, the findings of this study question the validity of using player self-reported throwing load as a marker of true throwing loads with most players recording in excess of 10 % error. High performance staff and players should consider whether the current accuracy of self-reported throwing load justifies the additional reporting burden on the players during training.

Nausea caused by exhaustive sprinting is associated with high lactate ([La-]) and hydrogen ion concentrations ([H+]) and fall in blood pCO2, thus raising the issue of whether there is a causal link between nausea and these variables. For this reason, this study aimed to determine whether interspersing repeated sprints (RS) with periods of active, compared with passive, recovery results in lower levels of both nausea and changes in [La-], [H+], and pCO2.Twelve male participants completed two separate sessions comprising four 30-s sprints separated by 20 min of either active (AR; cycling at 40% V˙O2peak) or passive recovery (PR). At 6 and 18 min of each recovery period, nausea was assessed via a visual analog scale (VAS), and blood samples were collected to measure [La-], [H+], and pCO2.RS significantly increased VAS score in both AR (P0.001) and PR (P0.01). After the first sprint, VAS was higher than preexercise in only AR (P0.01). AR was associated with lower VAS, [La-], [H+], and higher pCO2 (all P = 0.001) compared with PR after sprints 2-4. Linear mixed modeling indicated that each of the variables significantly predicts VAS scores (P0.0001). Repeated-measures correlation (rrm2) indicated that [La-] had the closest association with VAS (rrm2 = 0.22, P0.0001).The lower levels of both nausea and changes in [La-], [H+], and pCO2 in response to AR suggest that nausea associated with RS may be causally related with these variables. However, the absence of a close relationship between these variables after the first sprint and the findings that [La-], [H+], and pCO2 only account for 13%-22% of the variation in VAS indicate that other mechanisms may also mediate nausea.

Compounds [(LiI)1] and [(LiI)2]2 crystallize in the centrosymmetric space group P21/n. They are made up of neutral ligands [H2C(PPh2NSiMe3)2] (1) and [H2C(PPh2NSiMe3)(PPh2NH)] (2) and a LiI molecule. In both cases, N,N chelation with lithium is observed. Ligand 2 contains two different nitrogen centres viz., P=N(SiMe3) and P=N(H), which are coordinated unsymmetrically to lithium (Li–N = 2.055(8) and 2.072(8) A) to form [{LiI}{CH2(PPh2NSiMe3)×(PPh2NH)}] as monomer units that are linked via intermolecular coordination between NH and Li (2.097(8) A) to form a central four-membered ring, Li2N2 with four-coordinate lithium atoms. In contrast, [(LiI)1] is monomeric with a three-coordinate lithium centre. This disparity is reflected in the Li–I bond distances (2.699(11) A for [(LiI)1] and 2.824(7) A) for [(LiI)2]2). The dimer [(LiI)2]2 displays intramolecular Csp3H–π and intermolecular Csp2H–π interactions (between phosphorus-substituted phenyl groups).

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. The DNA binding affinity is weakened when the cognate ions Ni(II) and 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 noncognate 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 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 glutamic acid 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.

There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B 12 .

Nickel supports the growth of microbes from a variety of very different growth environments that affect nickel speciation. The mechanisms of nickel uptake and the molecular bases for the selectivity of this process are emerging. The recent surge of Ni-importer protein structures provides an understanding of Ni-recognition in the initial binding step of the import process. This review compares the structural basis for Ni-recognition in the complexes (ABC and ECF-type) that dominate primary (ATP-dependent) transport, with a focus on how the structures suggest mechanisms for Ni selectivity. The structures raise key questions about the mechanisms of nickel-transfer reactions involved in import. There is also a discussion of key experimental approaches necessary to help establish the physiological importance of these structures.

RcnR, a transcriptional regulator in Escherichia coli, derepresses the expression of the export proteins RcnAB upon binding Ni(II) or Co(II). Lack of structural information has precluded elucidation of the allosteric basis for the decreased DNA affinity in RcnR’s metal-bound states. Here, using hydrogen–deuterium exchange coupled with MS (HDX-MS), we probed the RcnR structure in the presence of DNA, the cognate metal ions Ni(II) and Co(II), or the noncognate metal ion Zn(II). We found that cognate metal binding altered the flexibility from the N-terminus through helix 1 and modulated the RcnR–DNA interaction. Apo-RcnR and RcnR–DNA complexes and the Zn(II)-RcnR complex exhibited similar 2H uptake kinetics, with fast-exchanging segments located in the N terminus, in helix 1 (residues 14–24), and at the C terminus. The largest difference in 2H incorporation between apo- and Ni(II)- and Co(II)-bound RcnR was observed in helix 1, which contains the N-terminus and His3, and has been associated with cognate metal binding. 2H uptake in helix 1 was suppressed in the Ni(II)- and Co(II)-bound RcnR complexes, in particular in the peptide corresponding to residues 14–24, containing Arg-14 and Lys-17. Substitution of these two residues drastically affected DNA-binding affinity, resulting in rcnA expression in the absence of metal. Our results suggest that cognate metal binding to RcnR orders its N-terminus, decreases helix 1 flexibility, and induces conformational changes that restrict DNA interactions with the positively charged residues Arg14 and Lys17. These metal-induced alterations decrease RcnR–DNA binding affinity, leading to rcnAB expression.

Summary Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilisation of methane through anaerobic methane oxidation. The enzyme employs an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a novel methyl radical/Ni(II)-thiolate intermediate. However, the biosynthesis of coenzyme F430 from the common primogenitor uroporphyrinoge III, incorporating 11 steric centres into the macrocycle, has remained poorly understood although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamisation and carbocyclic ring formation. We have now identified the proteins that catalyse coenzyme F430 biosynthesis from sirohydrochlorin, termed CfbA-E, and shown their activity. The research completes our understanding of how nature is able to construct its repertoire of tetrapyrrole-based life pigments, permitting the development of recombinant systems to utilise these metalloprosthetic groups more widely.

Nickel ions are required to cofactor several microbial enzymes. Transcriptionally regulated import and export systems have evolved to control cytoplasmic nickel levels and match physiological need with metal availability. In cases where there is no physiological need, only export systems will be required. Several different regulatory mechanisms have been identified that control gene expression in response to nickel ions. These include metal-responsive transcriptional regulators, periplasmic sensors that transmit Ni status across the inner membrane, and the recently identified NiCo riboswitch, the first RNA-based Ni sensor. The abundance of structural and functional studies available for these regulators provide an understanding of how nickel ions are selectivity recognized in the complex cellular milieu. Coordination chemistries that favour stable nickel ion binding over other metal ions enable proteins to couple conformational change to the presence of the correct metal. Interestingly, Ni-import systems are regulated by proteins that use four-coordinate planar geometry while export system regulators, both protein and RNA, favour six-coordinate sites, and as a result are also responsive to cobalt ions. The protein based sensors all have tight affinities for Ni, suggesting that intracellular buffered levels are well below a single atom/compartment volume. However, this leads to the unresolved question of how nickel ions are trafficked to metalloenzyme active sites in the face of a pool of high-affinity regulatory sites.

The metal affinities of metal-sensing transcriptional regulators co-vary with cellular metal concentrations over more than 12 orders of magnitude. To understand the cause of this relationship, we determined the structure of the Ni(II) sensor InrS and then created cyanobacteria (Synechocystis PCC 6803) in which transcription of genes encoding a Ni(II) exporter and a Ni(II) importer were controlled by InrS variants with weaker Ni(II) affinities. Variant strains were sensitive to elevated nickel and contained more nickel, but the increase was small compared with the change in Ni(II) affinity. All of the variant sensors retained the allosteric mechanism that inhibits DNA binding following metal binding, but a response to nickel in vivo was observed only when the sensitivity was set to respond in a relatively narrow (less than two orders of magnitude) range of nickel concentrations. Thus, the Ni(II) affinity of InrS is attuned to cellular metal concentrations rather than the converse.

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F

The RcnR metalloregulator represses the transcription of the Co(II) and Ni(II) exporter, RcnAB. Previous studies have shown that Co(II) and Ni(II) bind to RcnR in six-coordinate sites, resulting in derepression. Here, the roles of His60, His64, and His67 in specific metal recognition are examined. His60 and His64 correspond to ligands that are important for Cu(I) binding in the homologous Cu(I)-responsive metalloregulator, CsoR. These residues are known to be functionally important in RcnR transcriptional regulation. X-ray absorption spectroscopy (XAS) was used to examine the structure of bound cognate and noncognate metal ions, and lacZ reporter assays were used to assess the transcription of rcnA in response to metal binding in the three His → Cys mutations, H60C, H64C, and H67C. These studies confirm that both Ni(II) and Co(II) use His64 as a ligand. H64C-RcnR is also the only known mutant that retains a Co(II) response while eliminating the response to Ni(II) binding. XAS data indicate that His60 and His67 are potential Co(II) ligands. The effects of the mutations of His60, His64, and His67 on the structures of the noncognate metal ions [Zn(II) and Cu(I)] reveal that these residues have distinctive roles in binding noncognate metals. None of the His → Cys mutants in RcnR confer any response to Cu(I) binding, including H64C-RcnR, where the ligands involved in Cu(I) binding in CsoR are present. These data indicate that while the secondary, tertiary, and quaternary structures of CsoR and RcnR are quite similar, small changes in primary sequence reveal that the specific mechanisms involved in metal recognition are quite different.

Helicobacter pylori , a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys)(4) site to a Zn(His)(2)(Cys)(2) site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the beta-CH(2) protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys)(4)) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the flow of nickel traffic in the cell in response to nickel availability and pH.

Escherichia coli NikR, a repressor with homologs in other bacteria and archaea, was identified as a potential new member of the ribbon-helix-helix (beta-alpha-alpha) family of transcription factors in profile based sequence searches and in structure prediction experiments. Biophysical and biochemical characterization of the N-terminal domain of NikR show that it has many features expected of a beta-alpha-alpha protein including alpha-helical content, dimeric solution form, concentration dependent thermal stability, and ability to bind DNA in sequence-specific manner. Mutation of a residue predicted to be important for DNA-binding reduces operator affinity but does not affect the secondary structure or stability of the protein.

Escherichia coli NikR is a homotetrameric Ni 2+ - and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of two distinct domains. The N-terminal 50 amino acids of each chain forms part of the dimeric ribbon–helix–helix (RHH) domains, a well-studied DNA-binding fold. The 83-residue C-terminal nickel-binding domain forms an ACT (aspartokinase, chorismate mutase, and TyrA) fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni 2+ on DNA-binding activity. The molecular simulation data were analyzed using two different correlation measures based on fluctuations in atomic position and noncovalent contacts together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni 2+ - and DNA-binding sites, which are separated by 40 A. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains.

The nickel physiology of Escherichia coli is dominated by its Ni-Fe hydrogenase isozymes, which are expressed under anaerobic growth conditions. Hydrogenase activity in E. coli requires the NikABCDE nickel transporter, which is transcriptionally repressed by NikR in the presence of excess nickel. Recently, a nickel and cobalt-efflux protein, RcnA, was identified in E. coli. This study examines the effect of RcnA on nickel homeostasis in E. coli. Under nickel-limiting conditions, deletion of rcnA increased NikR activity in vivo. Nickel and cobalt-dependent regulation of rcnA expression required the newly identified transcriptional repressor RcnR (formerly YohL). Deletion of rcnR results in constitutive rcnA expression and a corresponding decrease in NikR activity. Purified RcnR binds directly to the rcnA promoter DNA fragment and this interaction is inhibited by nickel and cobalt. Nickel accumulation is affected differently among deletion strains with impaired nickel homeostasis. Surprisingly, in low nickel growth conditions rcnA expression is required for nickel import via NikABCDE. The data support a model with two distinct pools of nickel ions in E. coli. NikR bridges these two pools by controlling the levels of the hydrogenase-associated pool based on the nickel levels in the second pool.

Escherichia coli requires nickel under anaerobic growth conditions for the synthesis of catalytically active NiFe hydrogenases. Transcription of the NikABCDE nickel transporter, which is required for NiFe hydrogenase synthesis, was previously shown to be upregulated by FNR (fumarate-nit rate regulator) in the absence of oxygen and repressed by the NikR repressor in the presence of high extracellular nickel levels. We present here a detailed analysis of nikABCDE transcriptional regulation and show that it closely correlates with hydrogenase expression levels. We identify a nitrate-dependent mechanism for nikABCDE repression that is linked to the NarLX two-component system. NikR is functional under all nickel conditions tested, but its activity is modulated by the total nickel concentration present as well as by one or more components of the hydrogenase assembly pathway. Unexpectedly, NikR function is independent of NikABCDE function, suggesting that NikABCDE is a hydrogenase-specific nickel transporter, consistent with its original identification as a hydrogenase ( hyd ) mutant. Further, the results suggest that the hydrogenase assembly pathway is sequestered within the cell. A second nickel import pathway in E. coli is implicated in NikR function.

NikR is a metal-responsive transcription factor that controls nickel uptake in Escherichia coli by regulating expression of a nickel-specific ATP-binding cassette (ABC) transporter. We have determined the first two structures of NikR: the full-length apo repressor at a resolution of 2.3 A and the nickel-bound C-terminal regulatory domain at a resolution of 1.4 A. NikR is the only known metal-responsive member of the ribbon-helix-helix family of transcription factors, and its structure has a quaternary arrangement consisting of two dimeric DNA-binding domains separated by a tetrameric regulatory domain that binds nickel. The position of the C-terminal regulatory domain enforces a large spacing between the contacts that each NikR DNA-binding domain can make with the nik operator. The regulatory domain of NikR contains four nickel-binding sites at the tetramer interface, each exhibiting a novel square-planar coordination by three histidines and one cysteine side chain.

E. coli NikR repressor binds operator DNA in a nickel-dependent fashion. The pM affinity of NikR for nickel is mediated by its C-terminal 86 residues. Nickel binding induced additional secondary structure, decreased the compactness, and increased the stability of NikR. Tetramer formation by the C-terminal domain and intact NikR did not require nickel. High-affinity nickel binding decreased the NikR concentration needed to half maximally protect operator DNA from undetectable levels to 30 nM. The intracellular concentration of NikR in E. coli is high enough that saturation of the high-affinity nickel sites should lead to substantial occupancy of the nik operator. Nickel binding to a set of low-affinity NikR sites resulted in an additional large increase in operator affinity and substantially increased the size of the NikR footprint on the operator.

Nature 543, 78–82 (2017); doi:10.1038/nature21427 In this Article, we omitted to cite a relevant paper1 on coenzyme F430 biosynthesis, which identified the genes that encode the enzymes required for the transformation of sirohydrochlorin into coenzyme F430 and demonstrated their efficacy. Our study has confirmed these findings and also provided more detailed structural characterization of the pathway intermediates.

Cobalt and nickel play key roles in biological systems as cofactors in a small number of important enzymes. The majority of these are found in microbes. Evidence for direct roles for Ni(II) and Co(II) enzymes in higher organisms is limited, with the exception of the well-known requirement for the cobalt-containing vitamin B12 cofactor and the Ni-dependent urease in plants. Nonetheless, nickel in particular plays a key role in human health because of its essential role in microbes that inhabit various growth niches within the body. These roles can be beneficial, as can be seen with the anaerobic production and consumption of H2 in the digestive tract by bacteria and archaea that results in increased yields of short-chain fatty acids. In other cases, nickel has an established role in the establishment of pathogenic infection (Helicobacter pylori urease and colonization of the stomach). The synthesis of Co- and Ni-containing enzymes requires metal import from the extracellular milieu followed by the targeting of these metals to the appropriate protein and enzymes involved in metallocluster or cofactor biosynthesis. These metals are toxic in excess so their levels must be regulated carefully. This complex pathway of metalloenzyme synthesis and intracellular homeostasis requires proteins that can specifically recognize these metals in a hierarchical manner. This chapter focuses on quantitative and structural details of the cobalt and nickel binding sites in transport, trafficking and regulatory proteins involved in cobalt and nickel metabolism in microbes.

Escherichia coli actively imports nickel via the ATP-dependent NikABCDE permease. NikR, a protein of the ribbon-helix-helix family of transcription factors, represses expression of the nikABCDE operon in the presence of excessive concentrations of intracellular nickel. Here, the NikR operator site is identified within the nikABCDE promoter by footprinting and mutational analyses. The operator consists of two dyad-symmetric 5′-GTATGA-3′ recognition sequences separated by 16 base pairs. Mutations in the GTATGA sequences reduce NikR binding affinityin vitro and reduce repression of aP nik -lacZ fusion in vivo. Moreover, NikR is shown to be a direct sensor of nickel ions. Strong operator binding requires the continual presence of 20–50 μm nickel, indicating the presence of a low affinity nickel-binding site, and NikR dimers also contain two high affinity nickel-binding sites. In addition to both GTATGA sites and nickel, high affinity operator binding also requires the C-terminal domain of NikR.

The 2.2 Å crystalline structure of an oxidized active-site variant of Escherichia coli thioredoxin (Trx) has been solved. Trx is a 12 kDa enzyme which catalyzes the oxidation of dithiols and the reduction and isomerization of disulfides in other proteins. Its active site contains the common structural motif CXXC. Protein-disulfide isomerase (PDI), a 57 kDa homolog of Trx, contains four Trx-like domains. The three-dimensional structure of PDI is unknown. PDI-deficient Saccharomyces cerevisiae are inviable. An active-site variant of Trx which complements PDI-deficient yeast has the active-site sequence Cys32-Val33-Trp34-Cys35 (CVWC). The reduction potential of oxidized CVWC Trx (E°′ = −0.230 V) is altered significantly from that of the wild-type enzyme (E°′ = −0.270 V). However, the structure of the oxidized CVWC enzyme is almost identical to that of wild-type Trx. The addition of valine and tryptophan in the active site is likely to increase the reduction potential, largely by decreasing the pKa of the Cys32 thiol in the reduced enzyme. Unlike in wild-type Trx, significant protein–protein contacts occur in the crystal. Protein molecules related by a crystallographic twofold axis form a dimer in the crystal. The dimer forms as an extension of the twisted mixed β-sheet which composes the backbone of each Trx structure.

The active-site CXXC motif of thiol:disulfide oxidoreductases is essential for their catalysis of redox reactions. Changing the XX residues can perturb the reduction potential of the active-site disulfide bond of the Escherichia coli enzymes thioredoxin (Trx; CGPC) and DsbA (CPHC). The reduction potential is correlated with the acidity of the N-terminal cysteine residue of the CXXC motif. As the pKa is lowered, the disulfide bond becomes more easy to reduce. A change in pKa can account fully for a change in reduction potential in well-characterized CXXC motifs of DsbA but not of Trx. Formal analysis of the Nernst equation reveals that reduction potential contains both pH-dependent and pH-independent components. Indeed, the difference between the reduction potentials of wild-type Trx and wild-type DsbA cannot be explained solely by differences in thiol pKa values. Structural data for thiol:disulfide oxidoreductases reveal no single factor that determines the pH-independent component of the reduction potential. In addition, the pH-dependent component is complex when the redox state of the CXXC motif affects the titration of residues other than the thiols. These intricacies enable CXXC motifs to vary widely in their capacity to assist electron flow, and thereby engender a family of thiol:disulfide oxidoreductases that play diverse roles in biochemistry.

The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active-site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.

RcnR (resistance to cobalt and nickel regulator) is a 40 kDa homotetrameric protein and metalloregulator that controls the transcription of the Co(II) and Ni(II) exporter, RcnAB, by binding to DNA as an apo-protein and releasing DNA in response to specifically binding Co(II) and Ni(II) ions. Using x-ray absorption spectroscopy (XAS) to examine the structure of metals bound, and lacZ reporter assays of the transcription of RcnAB in response to metal binding, in WT- and mutant proteins, the roles of coordination number, ligand selection and residues in the N-terminus of the protein were examined as determinants in metal ion recognition. The studies show that the cognate metal ions, Co(II) and Ni(II), which bind in (N/O)5S six-coordinate sites, are distinguished from non-cognate metal ions (Cu(I) and Zn(II)), which bind only three protein ligands and one anion from the buffer, by coordination number and ligand selection. Using mutations of residues near the N-terminus, the N-terminal amine is shown to be a ligand of the cognate metal ions that is missing in the complexes with non-cognate metal ions. The side chain of His3 is also shown to play an important role in distinguishing metal ions. The imidazole group is shown to be a ligand in the Co(II) RcnR complex, but not in the Zn(II) complex. Further, His3 does not appear to bind to Ni(II), providing a structural basis for the differential regulation of RcnAB by the two cognate ions. The Zn(II) complexes change coordination number in response to the residue in position three. In H3C-RcnR, the Zn(II) complex is five-coordinate, and in H3E-RcnR the Zn(II) ion is bound to six protein ligands. The metric parameters of this unusual Zn(II) structure resemble those of the WT-Ni(II) complex, and the mutant protein is able to regulate expression of RcnAB in response to binding the non-cognate ion. The results are discussed within a protein allosteric model for gene regulation by metalloregulators.

Helicobacter pylori NikR (HpNikR) is a ribbon-helix-helix (RHH) DNA-binding protein that binds to several different promoter regions. The binding site sequences are not absolutely conserved. The ability of HpNikR to discriminate specific DNA sites resides partly in its nine-amino acid N-terminal arm. Previously, indirect evidence indicated that the arm exists in different conformations when HpNikR is bound to the nixA and ureA promoters. Here, we directly examined HpNikR conformation when it was bound to nixA and ureA DNA fragments by tethering (S)-1{[bis(carboxymethyl)amino]methyl}-2-{4-[(2-bromoacetyl)amino]phenylethyl}(carboxymethyl)amino]acetic acid, iron(III) to different positions in the N-terminal arm and RHH DNA binding domain. Different cleavage patterns at each promoter directly demonstrated that both the RHH domain and the arm adopt different conformations on the nixA and ureA promoters. Additionally, the two RHH domain dimers of the HpNikR tetramer are in distinct conformations at ureA. Site-directed mutagenesis identified an interchain salt bridge (Lys(48)-Glu(47')) in the RHH domain remote from the DNA binding interface that is required for high affinity binding to ureA but not nixA. Finally, DNA affinity measurements of wild-type HpNikR and a salt bridge mutant (K48A) to hybrid nixA-ureA promoters demonstrated that inverted repeat half-sites, spacers, and flanking DNA are all required for sequence-specific DNA binding by HpNikR. Notably, the spacer region made the largest contribution to DNA affinity. HpNikR exhibits a substantially expanded regulon compared with other NikR proteins. The results presented here provide a molecular basis for understanding regulatory network expansion by NikR as well as other prokaryotic regulatory proteins.

Metalloregulator function requires both sensitivity and selectivity to ensure metal-specific activity without interfering with intracellular metal trafficking pathways. Here, we examine the role of metal coordination geometry in the function of NikR and RcnR, two widely conserved nickel-responsive regulators that are both present in E. coli. The available data suggest an emerging trend in which coordination number is linked to metal-binding affinity, and thus regulatory function. The differences in coordination geometry also suggest that the kinetic mechanisms of metal-association and dissociation will contribute to metalloregulator function. We also discuss ways in which the ligand binding properties of metalloregulators may be tuned to alter the regulatory response.

The following systems have been investigated by 14N and (or)15N NMR spectroscopy: (a) the deprotonation of S7NH by n-BuLi, (b) solutions of S8, S7NH, 1,3- and 1,4-S6(NH)2, and K2SN2 in liquid ammonia, (c) the reaction of sodium azide with sulphur in hexamethylphosphoramide (HMPA), (d) the reactions of S7NH and S4(NH)4 with potassium amide in liquid ammonia in various molar ratios, and (e) the reaction of (NSCl)3 with potassium amide (1:6 molar ratio) in liquid ammonia. These investigations have provided an NMR characterization of the thermally unstable S7N− (δ15N ca. −324 ppm) and S2N2H− ions (δ14N + 7 and −149 ppm, 1J(14N−1H) 36 Hz, 1J(15N−H) 55 Hz, 2J(15N-15N) 2.2 Hz) in liquid ammonia, demonstrated the existence of S7N−, in addition to SSNSS−, as the major nitrogen-containing ingredient of sulphur–ammonia solutions (SAS), and confirmed the formation of the SSNSS−, but not S7N−, from the reaction of sodium azide and sulphur in HMPA. Consistent with previous chemical investigations, the NMR data show that the S7N− and S2N2H− ions are thermally unstable with respect to the formation of SSNSS− and S3N3−, respectively, in solution. Solutions of S4(NH)4/4KNH2 or (NSCl)3/6KNH2 show the same two 14N or 15N NMR resonances at ca. +97 and −108 ppm as a solution of NSN2− in liquid ammonia. Keywords: sulphur–nitrogen anions, nitrogen NMR, liquid ammonia.

Escherichia coli RcnR is a founding member of a recently discovered large and widespread structural family of bacterial transcription factors that are predicted to respond to a variety of environmental stresses. RcnR directly regulates transcription of the gene encoding the RcnA nickel and cobalt efflux protein by coordination of DNA-binding and metal-binding activities. A crystal structure of a Cu(I)-sensing homolog from Mycobacterium tuberculosis did not reveal how the novel all-α-helical fold of this protein family interacts with DNA because it lacks a well-characterized DNA-binding motif. In this study, we investigated the biophysical properties of the RcnR–DNA interaction using isothermal titration calorimetry and footprinting techniques. We found that an RcnR tetramer recognizes a TACT-G 6 -N-AGTA motif, of which there are two in the rcnA – rcnR intergenic region. G-tracts are found in many predicted binding sites of other RcnR/CsoR proteins, and here we show that they endow A-form DNA characteristics to the RcnR operator sites. Interestingly, RcnR also interacts nonspecifically with the ∼ 50 base pairs flanking the core binding site, resulting in DNA wrapping and the introduction of a single negative supercoil into plasmid DNA. Comparisons with other RcnR/CsoR proteins reveal likely key differences in DNA binding among members of this family that result from variations in the number and sequence of operator sites.

We examined the effects of urease and hydrogenase assembly gene deletions on NikR activation in H. pylori strains 26695 and G27. The loss of any component of urease assembly increased NikR activity under Ni 2+ -limiting conditions, as measured by reduced transcript levels and 63 Ni accumulation. Additionally, SlyD functioned in urease assembly in strain 26695.

Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E. coli, to provide a better understanding how different nickel levels are sensed in E. coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.

An enzyme linked immunosorbent assay (ELISA) was used to evaluate the prevalence and disease associations of antibodies to a range of negatively charged phospholipids in 111 patients with systemic lupus erythematosus (SLE). The frequency of one or more isotypes of different antiphospholipid antibodies (APLs) was similar (range 33%-45%). When individual isotypes were considered alone there was considerable variation (range 5%-32%). There were significant associations between thrombosis, thrombocytopenia, and central nervous system (CNS) disease but not abortion with elevated APL. Strong associations were found between raised anti-ds-DNA (Farr assay) and a positive direct Coomb's test with raised APL. Thus, APLs are common in SLE and are associated with discrete clinical and laboratory features. However, detection of antibodies to a range of negatively charged phospholipids added little clinically useful information to that obtained by measuring anticardiolipin antibody (ACL) alone. We cannot recommend the use of APLs other than ACL for routine testing.