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Biological environments present a complex array of metal-binding ligands. Metal-binding proteins have been the overwhelming focus of study because of their important and well-defined biological roles. Consequently, the presence of functional low molecular weight (LMW) metal-ligand complexes has been overlooked in terms of their roles in metallobiochemistry, particularly within cells. Recent studies in microbial systems have illuminated the different roles of L-histidine in nickel uptake, gene expression, and metalloenzyme maturation. In this focused critical review, these roles are surveyed in the context of the coordination chemistry of Ni(II) ions and the amino acid histidine, and the physico-chemical properties of nickel complexes of histidine. These complexes are fundamentally important to cellular metal homeostasis and further work is needed to fully define their contributions., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The importance of cellular low molecular weight ligands in metalloenzyme maturation is largely unexplored. Maturation of NiSOD requires post-translational N-terminal processing of the proenzyme, SodN, by its cognate protease, SodX. Here we provide evidence for the participation of L-histidine in the protease-dependent maturation of nickel-dependent superoxide dismutase (NiSOD) from Streptomyces coelicolor. In vitro studies using purified proteins cloned from S. coelicolor and overexpressed in E. coli support a model where a ternary complex formed between the substrate (SodN), the protease (SodX) and L-Histidine creates a novel Ni-binding site that is capable of the N-terminal processing of SodN and specifically incorporates Ni into the apo-NiSOD product. Thus, L-Histidine serves many of the functions associated with a metallochaperone or, conversely, eliminates the need for a metallochaperone in NiSOD maturation., (© The Author(s) 2023. Published by Oxford University Press.)

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 5 th 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., Trial Registration: PROSPERO (Registration number: CRD42021251080)., (© 2023. The Author(s).)

Two resins with the hydroxamic acid siderophore desferrioxamine B (DFOB) immobilised as a free ligand or its Fe(iii) complex were prepared to screen the Streptomyces pilosus proteome for proteins involved in siderophore-mediated Fe(iii) uptake. The resin design included a disulfide bond to enable the release of bound proteins under mild reducing conditions. Proteomics analysis of the bound fractions did not identify proteins associated with siderophore-mediated Fe(iii) uptake, but identified nickel superoxide dismutase (NiSOD), which was enriched on the apo-DFOB-resin but not the Fe(iii)-DFOB-resin or the control resin. While DFOB is unable to sequester Fe(iii) from sites deeply buried in metalloproteins, the coordinatively unsaturated Ni(ii) ion in NiSOD is present in a surface-exposed loop region at the N-terminus, which might enable partial chelation. The results were consistent with the notion that the apo-DFOB-resin formed a ternary complex with NiSOD, which was not possible for either the coordinatively saturated Fe(iii)-DFOB-resin or the non-coordinating control resin systems. In support, ESI-TOF-MS measurements from a solution of a model Ni(ii)-SOD peptide and DFOB showed signals that correlated with a ternary Ni(ii)-SOD peptide-DFOB complex. Although any biological implications of a DFOB-NiSOD complex are unclear, the work shows that the metal coordination properties of siderophores might influence an array of metal-dependent biological processes beyond those established in iron uptake., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Objectives: 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., Design: Cross-sectional cohort study., Methods: 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., Results: 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 %., Conclusions: 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., (Copyright © 2022 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

Inorganic metals supplement the chemical repertoire of organic molecules, especially proteins. This requires the correct metals to associate with proteins at metalation. Protein mismetalation typically occurs when excesses of unbound metals compete for a binding site ex vivo. However, in biology, excesses of metal-binding sites typically compete for limiting amounts of exchangeable metals. Here, we summarise mechanisms of metal homeostasis that sustain optimal metal availabilities in biology. We describe recent progress to understand metalation by comparing the strength of metal binding to a protein versus the strength of binding to competing sites inside cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The management of procedural pain in infants is suboptimal, in part, compounded by the scarcity of a simple, accurate, and reliable method of assessing such pain. In this study, we aimed to evaluate the psychometric properties of the PainChek Infant, a point-of-care mobile application that uses automated facial evaluation and analysis in the assessment of procedural pain in infants., Methods: Video recordings of 40 infants were randomly chosen from a purposely assembled digital library of 410 children undergoing immunisation as part of their standard care in Prishtina, Kosovo, between April 4, 2017, and July 11, 2018. For each infant recording, four 10 s video segments were extracted, corresponding to baseline, vaccine preparation, during vaccination, and recovery. Four trained assessors did pain assessments on the video segments of 30 infants, using PainChek Infant standard, PainChek Infant adaptive, the Neonatal Facial Coding System-Revised (NFCS-R) single, the NFCS-R multiple, and the Observer administered Visual Analogue Scale (ObsVAS), on two separate occasions. PainChek Infant's performance was compared to NFCS-R and ObsVAS using correlation in changes in pain scores, intra-rater and inter-rater reliability, and internal consistency., Findings: 4303 pain assessments were completed in two separate testing sessions, on Aug 31, and Oct 19, 2020. The study involved videos of 40 infants aged 2·2-6·9 months (median age 3·4 months [IQR 2·3-4·5]). All pain assessment tools showed significant changes in the recorded pain scores across the four video segments (p≤0·0006). All tools were found to be responsive to procedure-induced pain, with the degree of change in pain scores not influenced by pre-vaccination pain levels. PainChek Infant pain scores showed good correlation with NFCS-R and ObsVAS scores (r=0·82-0·88; p<0·0001). PainChek Infant also showed good to excellent inter-rater reliability (ICC=0·81-0·97, p<0·001) and high levels of internal consistency (α=0·82-0·97)., Interpretation: PainChek Infant's use of automated facial expression analysis could offer a valid and reliable means of assessing and monitoring procedural pain in infants. Its clinical utility in clinical practice requires further research., Funding: PainChek., Competing Interests: Declaration of interests KH and JDH are shareholders in PainChek (formerly known as EPAT Technologies), which is commercialising the PainChek Infant. They are also named as coinventors with Mustafa Atee on the patent entitled a pain assessment method and system (patent granted in Australia [AU2015306075B2], Japan [JP6657216B2], USA [US10398372B2], and China [CN106572820A]. Patent pending from Europe [EP3182893A4], and from the World Intellectual Property Organization [WO2016025989A1]). KH is employed as a consultant by PainChek, while also serving as an associate Professor at the University of Prishtina, Kosovo and university associate at the Curtin Medical School, Curtin University, WA, Australia. JDH is employed as the chief scientific officer of PainChek and holds an adjunct Professor appointment in the Curtin Medical School, Curtin University, WA, Australia. PTC was engaged through DATaR Consulting and was paid as an independent private consultant to undertake the biostatistical analysis for the project by PainChek. PTC also holds adjunct appointments at the Institute for Health Research, the University of Notre Dame Australia, Fremantle, WA, Australia, and School of Medical and Health Sciences, Edith Cowan University, Joondalup, WA, Australia., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Introduction: 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., Methods: 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., Results: RS significantly increased VAS score in both AR (P < 0.001) and PR (P < 0.01). After the first sprint, VAS was higher than preexercise in only AR (P < 0.01). 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 (P < 0.0001). Repeated-measures correlation (rrm2) indicated that [La-] had the closest association with VAS (rrm2 = 0.22, P < 0.0001)., Conclusion: 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., (Copyright © 2021 by the American College of Sports Medicine.)

Objectives: To explore the association between depressive symptoms and recent head-related trauma (diagnosed concussion, subconcussive impacts) in semiprofessional male Australian Football (AF) players., Methods: Sixty-nine semiprofessional male players from a West Australian Football League (WAFL) club participated in the study (M age =21.81, SD=2.91 years). Depressive symptoms were measured using the Centre for Epidemiological Studies Depression Scale. Injuries and potential confounding variables (eg, pre-existing mental health condition; alcohol or drug hangovers; experiencing a stressful event) were self-reported anonymously using the WAFL Injury Report Survey. Both tools were administered every 2-weeks over the first 22-weeks of the WAFL season. Controlling for potential confounding variables and other injuries, a repeated measures generalised estimating equations model assessed the risk of clinically relevant depressive symptoms occurring, when diagnosed concussion or subconcussive impacts were experienced., Results: A total of 10 concussions and 183 subconcussive impacts were reported. Players who experienced a concussion were almost nine times more likely to experience clinically relevant depressive symptoms (OR 8.88, 95% CI 2.65 to 29.77, p<0.001). Although elevated, depressive symptoms following subconcussive impacts were not statistically significant (OR 1.13, 95% CI 0.67 to 1.92, p=0.641)., Conclusion: These findings indicate that semiprofessional AF athletes may be at risk of experiencing depressive symptoms after concussion. Severity (concussion vs subconcussive impacts) and dose (number of impacts) appear to have an important relationship with depressive symptom outcomes in this cohort and should be considered for further research and management of player welfare., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

This study aims to determine whether there is a relationship between nausea level and lactic acidosis during recovery from sprinting. In all, 13 recreationally active males completed a 60 s bout of maximal intensity cycling. Prior to and for 45 min following exercise, blood pH, pCO 2 , and lactate levels were measured together with nausea. In response to sprinting, nausea, lactate, and H + concentrations increased and remained elevated for at least 10 min (p < .001), whereas pCO 2 increased only transiently (p < .001) before falling below pre-exercise levels (p < .001), with all these variables returning toward pre-exercise levels during recovery. Both measures of nausea adopted for analyses (nausea profile, NP; visual analogue scale, VAS), demonstrated significant repeated measures correlation (rmcorr) post-exercise between nausea and plasma lactate (VAS and NPr rm > 0.595, p < .0001) and H + concentrations (VAS and NPr rm > 0.689, p < .0001), but an inconsistent relationship with pCO 2 (VAS r rm  = 0.250, p = .040; NP r rm  = 0.144, p = .248) and bicarbonate levels (VAS r rm  = -0.252, p = .095; NP r rm  = -0.397, p = .008). Linear mixed modeling was used to predict the trajectory of nausea over time, with both lactate and H + concentrations found to be key predictors of nausea (p < .0001). In conclusion, this study reveals a strong positive relationship between nausea and both H + and lactate concentrations during recovery from sprinting, a finding consistent with H + and lactate being potential mediators of nausea post-sprinting. However, as the timing of the recovery of both H + and lactate was delayed, compared to that of nausea, further research is required to confirm these findings and investigate other potential mechanisms., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

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 .

Background: Natural genetic variation contributes towards athletic performance in various strength/power and endurance based sports. To date, no studies have explored the genetic predisposition towards skill and athletic performance in Australian Football (AF) players., Methods: The present pilot study recruited 30 sub-elite AF players who completed tests of endurance, power and technical skill. Specific polymorphisms in nine genes were screened, and assessed for a possible association with athletic and skill traits., Results: Statistical analysis using generalized linear models identified a number of polymorphisms predictive of endurance and technical skill. The angiotensin-converting enzyme (ACE), normally responsible for regulation of body fluid volume, was a significant factor in predicting 'all round' athletic performance and skill. Specifically, the deletion allele (DD) of ACE was identified as a predictor for AF power (P≤0.008), endurance (P=0.001) and skill assessments (P≤0.003). In addition, polymorphisms in the brain-derived neurotrophic factor, D2 dopamine receptor, and catechol-O-methyltransferase genes were also shown to contribute to kicking skill outcomes (P≤0.044)., Conclusions: This is the first study to implicate the ACE deletion allele for a multidimensional sport in such a way. Further, the results from this study have identified several new candidate genes in predicting athletic and technical skill outcomes.

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 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 2 H 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 2 H incorporation between apo- and Ni(II)- and Co(II)-bound RcnR was observed in helix 1, which contains the N terminus and His-3, and has been associated with cognate metal binding. 2 H 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 Arg-14 and Lys-17. These metal-induced alterations decrease RcnR-DNA binding affinity, leading to rcnAB expression., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

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 P RcnAB . 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) 5 S 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 P rcnA 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.

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 430 , a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme F 430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F 430 from sirohydrochlorin, termed CfbA-CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.

Most organisms are exposed to the genotoxic chemical formaldehyde, either from endogenous or environmental sources. Therefore, biology has evolved systems to perceive and detoxify formaldehyde. The frmRA(B) operon that is present in many bacteria represents one such system. The FrmR protein is a transcriptional repressor that is specifically inactivated in the presence of formaldehyde, permitting expression of the formaldehyde detoxification machinery (FrmA and FrmB, when the latter is present). The X-ray structure of the formaldehyde-treated Escherichia coli FrmR (EcFrmR) protein reveals the formation of methylene bridges that link adjacent Pro2 and Cys35 residues in the EcFrmR tetramer. Methylene bridge formation has profound effects on the pattern of surface charge of EcFrmR and combined with biochemical/biophysical data suggests a mechanistic model for formaldehyde-sensing and derepression of frmRA(B) expression in numerous bacterial species.

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.

Introduction: Systematic reviews suggest that a longer duration of breast-feeding is associated with a reduction in the risk of later overweight and obesity. Most studies examining breast-feeding in relation to adiposity have not used longitudinal analysis. In our study, we aimed to examine early infant feeding and adiposity risk in a longitudinal cohort from birth to young adulthood using new as well as published data., Methods: Data from the Western Australian Pregnancy Cohort (Raine) Study in Perth, W.A., Australia, were used to examine associations between breast-feeding and measures of adiposity at 1, 2, 3, 6, 8, 10, 14, 17, and 20 years., Results: Breast-feeding was measured in a number of ways. Longer breast-feeding (in months) was associated with reductions in weight z-scores between birth and 1 year (β = -0.027; p < 0.001) in the adjusted analysis. At 3 years, breast-feeding for <4 months increased the odds of infants experiencing early rapid growth (OR 2.05; 95% CI 1.43-2.94; p < 0.001). From 1 to 8 years, children breast-fed for ≤4 months compared to ≥12 months had a significantly greater probability of exceeding the 95th percentile of weight. The age at which breast-feeding was stopped and a milk other than breast milk was introduced (introduction of formula milk) played a significant role in the trajectory of the BMI from birth to 14 years; the 4-month cutoff point was consistently associated with a higher BMI trajectory. Introduction of a milk other than breast milk before 6 months compared to at 6 months or later was a risk factor for being overweight or obese at 20 years of age (OR 1.47; 95% CI 1.12-1.93; p = 0.005)., Discussion: Breast-feeding until 6 months of age and beyond should be encouraged and is recommended for protection against increased adiposity in childhood, adolescence, and young adulthood. Adverse long-term effects of early growth acceleration are fundamental in later overweight and obesity. Formula feeding stimulates a higher postnatal growth velocity, whereas breast-feeding promotes slower growth and a reduced likelihood of overweight and obesity. Biological mechanisms underlying the protective effect of breast-feeding against obesity are based on the unique composition and metabolic and physiological responses to human milk., (© 2014 S. Karger AG, Basel.)

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.

Nickel is an important cofactor for several microbial enzymes. The ATP-dependent NikABCDE transporter is one of several types of uptake pathways known to be important for nickel acquisition in microbes. The Escherichia coli NikA periplasmic binding protein is structurally homologous to the di- and oligopeptide binding proteins, DppA and OppA. This structural similarity raises interesting questions regarding the evolutionary relationships between the recognition of nickel ions and short peptides. We find that in defined minimal growth medium NikABCDE transports nickel ions in the presence of exogenously added L-histidine (L-His), but not D-histidine. Both nickel uptake in cells and nickel binding to purified NikA showed an L-His concentration dependence consistent with recognition of a Ni-(L-His)₂ complex. This discovery reveals parallels to the transport of other metal complexes, notably iron, and suggests the structural diversity of nickel transporters may arise from the need to recognize extracellular nickel complexed with different organic ligands, whether they be exogenously or endogenously produced. Further, these results suggest that experiments examining the physiology and ecology of nickel-requiring microbes should account for the possibility that the growth medium may not support nickel uptake.

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 apoprotein 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 RcnA 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)(5)S 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.

To examine the strength and direction of the relationship between physical activity level, screen use and BMI in a cohort at ages 6, 8, 10 and 14 yrs as part of a prospective longitudinal cohort study. The sample comprised 1403 males and females who participated in the follow-up survey at 14 yrs of age between 2003 and 2005. Exploratory structural equation modelling was used to examine the interrelationships between physical activity level, BMI and screen time at 6, 8, 10 and 14 yrs. Predictors of BMI at 6, 8, 10 and 14 yrs explained 1.3, 76.1, 80.1 and 73.1 percent of the variances, respectively, with previous BMI the largest predictor [χ(2)=43.082, df=36, p=194]. Increased screen time predicted higher BMI and lower physical activity at 8 and 10 yrs but not 14 yrs. At 14 yrs, physical activity predicted BMI. Sedentary patterns of behaviour in early childhood were predictive of later and concurrent obesity, whereas physical activity was predictive of obesity in adolescence. Different intervention targets are required for children and adolescents., (Copyright © 2011 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

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.

Background: Helicobacter pylori's Fur regulatory protein controls transcription of dozens of genes in response to iron availability, acidity and oxidative stress, and affects the vigor of infection and severity of disease. It is unusual among Fur family proteins in being active both when iron-loaded and iron-free. METHOLODOLGY/PRINCIPAL FINDINGS: We tested if H. pylori fur mutations could affect resistance to metronidazole (Mtz), an anti-H. pylori prodrug rendered bactericidal by chemical reduction. Point mutations were made by PCR in DNA containing fur and a downstream chloramphenicol resistance gene, and were placed in the H. pylori chromosome by transformation of a fur-deletion (Δfur) strain. Several substitutions affecting H. pylori Fur's ∼10 residue N terminal arm, which has no counterpart in prototype (E. coli-type) Fur proteins, increased Mtz resistance, as did mutations affecting the region between DNA binding and dimerization domains. Three types of mutations decreased resistance more than did Δfur: substitutions affecting the N-terminal arm; substitutions affecting the metal binding pocket; and nonsense mutations that resulted in a truncated Fur protein with no C-terminal dimerization domain. Most metal binding pocket mutations were obtained only in fur genes with additional inactivating mutations, and thus seemed deleterious or lethal because they., Conclusions/significance: These results establish that H. pylori Fur's distinctive N terminal arm is functional, and more generally illustrate that point mutations can confer informative phenotypes, distinct from those conferred by null mutations. We propose that fur mutations can affect Mtz susceptibility by altering the balance among Fur's several competing activities, and thereby the expression of genes that control cellular redox potential or elimination of bactericidal Mtz activation products. Further analyses of selected mutants should provide insights into Fur interactions with other cellular components, metabolic circuitry, and how H. pylori thrives in its special gastric niche.

NikR is a nickel-responsive ribbon-helix-helix transcription factor present in many bacteria and archaea. The DNA binding properties of Escherichia coli and Helicobacter pylori NikR (factors EcNikR and HpNikR, respectively) have revealed variable features of DNA recognition. EcNikR represses a single operon by binding to a perfect inverted repeat sequence, whereas HpNikR binds to promoters from multiple genes that contain poorly conserved inverted repeats. These differences are due in large part to variations in the amino acid sequences of the DNA-contacting beta-sheets, as well as residues preceding the beta-sheets of these two proteins. We present here evidence of another variation in DNA recognition by the NikR protein from Geobacter uraniireducens (GuNikR). GuNikR has an Arg-Gly-Ser beta-sheet that binds specifically to an inverted repeat sequence distinct from those recognized by Ec- or HpNikR. The N-terminal residues that precede the GuNikR beta-sheet residues are required for high-affinity DNA binding. Mutation of individual arm residues dramatically reduced the affinity of GuNikR for specific DNA. Interestingly, GuNikR tetramers are capable of binding cooperatively to the promoter regions of two different genes, nik(MN)1 and nik(MN)2. Cooperativity was not observed for the closely related G. bemidjiensis NikR, which recognizes the same operator sequence. The cooperative mode of DNA binding displayed by GuNikR could affect the sensitivity of transporter gene expression to changes in intracellular nickel levels.

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.

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.

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-alpha-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 approximately 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 Ni2+-limiting conditions, as measured by reduced transcript levels and 63Ni 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.

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 Ni(2+)-dependent transcription factor NikR is widespread among microbes. The two experimentally characterized NikR orthologs, from Helicobacter pylori and Escherichia coli, display vastly different regulatory capabilities in response to increased intracellular Ni(2+). Here, we demonstrate that the nine-residue N-terminal arm present in H. pylori NikR plays a critical role in the expanded regulatory capabilities of this NikR family member. Specifically, the N-terminal arm is required to inhibit NikR binding to low affinity and nonspecific DNA sequences and is also linked to a cation requirement for NikR binding to the nixA promoter. Site-directed mutagenesis and arm-truncation variants of NikR indicate that two residues, Asp-7 and Asp-8, are linked to the cation requirement for binding. Pro-4 and Lys-6 are required for maximal DNA binding affinity of the full-length protein to both the nixA and ureA promoters. The N-terminal arm is highly variable among NikR family members, and these results suggest that it is an adaptable structural feature that can tune the regulatory capabilities of NikR to the nickel physiology of the microbe in which it is found.

Studies of the transcriptional repression of the Ni-specific permease encoded by the Pnik operon by Escherichia coli NikR using a LacZ reporter assay establish that the NikR response is specific to nickel in vivo. Toward understanding this metal ion-specific response, X-ray absorption spectroscopy (XAS) analysis of various M-NikR complexes (M = Co(II), Ni(II), Cu(II), Cu(I), and Zn(II)) was used to show that each high-affinity binding site metal adopts a unique structure, with Ni(II) and Cu(II) being the only two metal ions to feature planar four-coordinate complexes. The results are consistent with an allosteric mechanism whereby the geometry and ligand selection of the metal present in the high-affinity site induce a unique conformation in NikR that subsequently influences DNA binding. The influence of the high-affinity metal on protein structure was examined using hydrogen/deuterium (H/D) exchange detected by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Each NikR complex gives rise to differing amounts of H/D exchange; Zn(II)- and Co(II)-NikR are most like apo-NikR, while the exchange time course is substantially different for Ni(II) and to a lesser extent for Cu(II). In addition to the high-affinity metal binding site, E. coli NikR has a low-affinity metal-binding site that affects DNA binding affinity. We have characterized this low-affinity site using XAS in heterobimetallic complexes of NikR. When Cu(II) occupies the high-affinity site and Ni(II) occupies the low-affinity site, the Ni K-edge XAS spectra show that the Ni site is composed of six N/O-donors. A similar low-affinity site structure is found for the NikR complex when Co(II) occupies the low-affinity site and Ni(II) occupies the high-affinity site, except that one of the Co(II) ligands is a chloride derived from the buffer.

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.

The fit locus, encoding two proteins, FitA and FitB, was identified in a genetic screen for Neisseria gonorrhoeae determinants that affect trafficking across polarized epithelial cells. To better understand how the locus may control these activities, we have undertaken a biochemical analysis of FitA and FitB. FitA is a DNA-binding protein with a putative ribbon-helix-helix (RHH) motif. Purified FitA forms a homodimer that binds a 150 bp fit promoter sequence containing the translational start site. A putative beta strand mutant of FitA, FitA(R7A), is unable to bind this DNA, supporting further that FitA is a RHH protein. FitB interacts with FitA to form a 98 kDa complex. FitA/B binds DNA with a 38-fold higher affinity than the FitA homodimer. In DNase I footprint assays, FitA/B protects a 62-bp region within the fit promoter containing the predicted -10 sequence and an 8-bp inverted repeat, TGCTATCA-N(12)-TGATAGCA. FitA/B(His) is able to bind to either half-site alone with high affinity.

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.

The Pyrococcus horikoshii OT3 genome contains a gene (PH0601 or nikR) encoding a protein (PhNikR) that shares 33.8% amino acid sequence identity with Escherichia coli nickel responsive repressor NikR (EcNikR), including many residues that are functionally important in the E.coli ortholog. We succeeded in crystallization and structural characterization of PhNikR in the apo form and two nickel bound forms that exhibit different conformations, open and closed. Moreover, we have identified a putative "low-affinity" nickel-binding pocket in the closed form. This binding site has unusual nickel coordination and exhibits high sensitivity to phosphate in the crystal structure. Analysis of the PhNikR structures and structure-based mutational studies with EcNikR reveals a plausible mechanism of nickel-dependent promoter recognition by the NikR family of proteins.

Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.

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.

The uptake of nickel in Escherichia coli and other microorganisms is transcriptionally regulated by the NikR repressor or its homologs. Here we report the structure of the high-affinity nickel-binding site in NikR and show that it responds dramatically to DNA binding. X-ray absorption spectroscopy reveals that nickel in the holo-NikR protein is bound in a novel four-coordinate planar site consisting of two histidines, one additional O- or N-donor ligand and one S-donor ligand. Site-directed mutation of His87, His89, Cys95 or Glu97 in NikR to alanine eliminates high-affinity nickel binding and abolishes DNA binding but maintains stable protein folding. An unanticipated feature of the NikR structure is that the nickel coordination responds to DNA binding. A six-coordinate nickel site composed of O- or N-donor ligands, but lacking cysteine, forms when NikR binds to operator DNA. Because nickel binding and DNA binding are mediated by different domains within NikR, a communication link between the two domains is implicated, consistent with the finding that the nickel-binding site in a fragment corresponding to the C-terminal domain of NikR is structurally distinct from that found in holo-NikR.

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.

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 affinity in vitro and reduce repression of a P(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 micrometer 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.

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.

The 2.2 A 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 degrees ' = -0.230 V) is altered significantly from that of the wild-type enzyme (E degrees ' = -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 pK(a) 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 beta-sheet which composes the backbone of each Trx structure.