Evaluation of the regulatory model for Ni2+ sensing by Nur from Streptomyces coelicolor.

Streptomyces coelicolor is a soil-dwelling bacterium that is medically important due to its ability to produce several antibiotics, and nickel accumulation within this organism has been shown to prevent the production of the antibiotic undecylprodigiosin. The transcriptional repressor important in regulation of nickel uptake is the homodimeric Nur, a member of the Fur family. Nur contains two metal-binding sites per monomer: the M-site and the Ni-site. The work described here seeks to determine the roles of each of the metal-binding sites to establish a model of Nur activity through mutational studies, metal titrations, and fluorescence anisotropy. Through these studies, a model of Nur activity is proposed in which femtomolar metal binding to one M-site of Nur prompts DNA-binding, and metal binding to the second M-site fully activates the protein. Evidence is provided that shows cooperative metal binding to the Ni-site, but this process dampens affinity for promoter DNA.

In depth, thermodynamic analysis of Ca2+ binding to human cardiac troponin C: Extracting buffer-independent binding parameters.

BACKGROUND: Characterizing the thermodynamic parameters behind metal-biomolecule interactions is fundamental to understanding the roles metal ions play in biology. Isothermal Titration Calorimetry (ITC) is a "gold-standard" for obtaining these data. However, in addition to metal-protein binding, additional equilibria such as metal-buffer interactions must be taken into consideration prior to making meaningful comparisons between metal-binding systems. METHODS: In this study, the thermodynamics of Ca2+ binding to three buffers (Bis-Tris, MES, and MOPS) were obtained from Ca2+-EDTA titrations using ITC. These data were used to extract buffer-independent parameters for Ca2+ binding to human cardiac troponin C (hcTnC), an EF-hand containing protein required for heart muscle contraction. RESULTS: The number of protons released upon Ca2+ binding to the C- and N-domain of hcTnC were found to be 1.1 and 1.2, respectively. These values permitted determination of buffer-independent thermodynamic parameters of Ca2+-hcTnC binding, and the extracted data agreed well among the buffers tested. Both buffer and pH-adjusted parameters were determined for Ca2+ binding to the N-domain of hcTnC and revealed that Ca2+ binding under aqueous conditions and physiological ionic strength is both thermodynamically favorable and driven by entropy. CONCLUSIONS: Taken together, the consistency of these data between buffer systems and the similarity between theoretical and experimental proton release is indicative of the reliability of the method used and the importance of extracting metal-buffer interactions in these studies. GENERAL SIGNIFICANCE: The experimental approach described herein is clearly applicable to other metal ions and other EF-hand protein systems.

Conserved cysteine residues are necessary for nickel-induced allosteric regulation of the metalloregulatory protein YqjI (NfeR) in E. coli.

Transition metal homeostasis is necessary to sustain life. First row transition metals act as cofactors within the cell, performing vital functions ranging from DNA repair to respiration. However, intracellular metal concentrations exceeding physiological requirements may be toxic. In E. coli, the YqjH flavoprotein is thought to play a role in iron homeostasis. YqjH is transcriptionally regulated by the ferric uptake regulator and a newly discovered regulator encoded by yqjI. The apo-form of YqjI is a transcriptional repressor of both the yqjH and yqjI genes. YqjI repressor function is disrupted upon binding of nickel. The YqjI N-terminus is homologous to nickel-binding proteins, implicating this region as a nickel-binding domain. Based on function, yqjI and yqjH should be renamed Ni-responsive Fe-uptake regulator (nfeR) and Ni-responsive Fe-uptake flavoprotein (nfeF), respectively. X-ray Absorption Spectroscopy was employed to characterize the nickel binding site(s) within YqjI. Putative nickel binding ligands were targeted by site-directed mutagenesis and resulting variants were analyzed in vivo for repressor function. Isothermal titration calorimetry and competitive binding assays were used to further quantify nickel interactions with wild-type YqjI and its mutant derivatives. Results indicate plasticity in the nickel binding domain of YqjI. Residues C42 and C43 were found to be required for in vivo response of YqjI to nickel stress, though these residues are not required for in vitro nickel binding. We propose that YqjI may contain a vicinal disulfide bond between C42 and C43 that is important for nickel-responsive allosteric interactions between YqjI domains.

Dissecting ITC data of metal ions binding to ligands and proteins.

BACKGROUND: ITC is a powerful technique that can reliably assess the thermodynamic underpinnings of a wide range of binding events. When metal ions are involved, complications arise in evaluating the data due to unavoidable solution chemistry that includes metal speciation and a variety of linked equilibria. SCOPE OF REVIEW: This paper identifies these concerns, provides recommendations to avoid common mistakes, and guides the reader through the mathematical treatment of ITC data to arrive at a set of thermodynamic state functions that describe identical chemical events and, ideally, are independent of solution conditions. Further, common metal chromophores used in biological metal sensing studies are proposed as a robust system to determine unknown solution competition. MAJOR CONCLUSIONS: Metal ions present several complications in ITC experiments. This review presents strategies to avoid these pitfalls and proposes and experimentally validates mathematical approaches to deconvolute complex equilibria that exist in these systems. GENERAL SIGNIFICANCE: This review discusses the wide range of complications that exists in metal-based ITC experiments. It provides a starting point for scientists new to this field and articulates concerns that will help experienced researchers troubleshoot experiments.

Stabilization of Cu(I) for binding and calorimetric measurements in aqueous solution.

Conditions have been developed for the comproportionation reaction of Cu(2+) and copper metal to prepare aqueous solutions of Cu(+) that are stabilized from disproportionation by MeCN and other Cu(+)-stabilizing ligands. These solutions were then used in ITC measurements to quantify the thermodynamics of formation of a set of Cu(+) complexes (Cu(I)(MeCN)3(+), Cu(I)Me6Trien(+), Cu(I)(BCA)2(3-), Cu(I)(BCS)2(3-)), which have stabilities ranging over 15 orders of magnitude, for their use in binding and calorimetric measurements of Cu(+) interaction with proteins and other biological macromolecules. These complexes were then used to determine the stability and thermodynamics of formation of a 1 : 1 complex of Cu(+) with the biologically important tri-peptide glutathione, GSH. These results identify Me6Trien as an attractive Cu(+)-stabilizing ligand for calorimetric experiments, and suggest that caution should be used with MeCN to stabilize Cu(+) due to its potential for participating in unquantifiable ternary interactions.

Allosteric inhibition of a zinc-sensing transcriptional repressor: insights into the arsenic repressor (ArsR) family.

The molecular basis of allosteric regulation remains a subject of intense interest. Staphylococcus aureus CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of bacterial homodimeric metal-sensing proteins and has emerged as a model system for understanding allosteric regulation of operator DNA binding by transition metal ions. Using unnatural amino acid substitution and a standard linkage analysis, we show that a His97' NH(ε2)...O=C His67 quaternary structural hydrogen bond is an energetically significant contributor to the magnitude of the allosteric coupling free energy, ∆Gc. A "cavity" introduced just beneath this hydrogen bond in V66A/L68V CzrA results in a significant reduction in regulation by Zn(II) despite adopting a wild-type global structure and Zn(II) binding and DNA binding affinities only minimally affected from wild type. The energetics of Zn(II) binding and heterotropic coupling free energies (∆Hc, -T∆Sc) of the double mutant are also radically altered and suggest that increased internal dynamics leads to poorer allosteric negative regulation in V66A/L68V CzrA. A statistical coupling analysis of 3000 ArsR proteins reveals a sector that links the DNA-binding determinants and the α5 Zn(II)-sensing sites through V66/L68 in CzrA. We propose that distinct regulatory sites uniquely characteristic of individual ArsR proteins result from evolution of distinct connectivities to this sector, each capable of driving the same biological outcome, transcriptional derepression.

Illuminating allostery in metal sensing transcriptional regulators.

The intracellular availability of all biologically required transition metal ions in bacteria, e.g., Zn, Cu, Fe, as well as the detoxification of nonbiological heavy metal pollutants, is controlled at the molecular level by a panel of metalloregulatory or "metal sensor" proteins. Metal sensor proteins are specialized allosteric proteins that regulate the transcription of genes linked to transition metal homeostasis as a result of direct binding of a single metal ion or two closely related metal ions, to the exclusion of all others. In many cases, the binding of the cognate metal ion induces a structural change in a metal sensor oligomer that either activates or inhibits operator DNA binding. A quantitative measure of the degree to which a particular metal drives metalloregulation of transcription is the allosteric coupling-free energy, ΔG(c). In this chapter, we outline detailed spectroscopically derived methods for measuring metal binding affinity, K(Me), as well as ΔG(c) independent of K(Me), presented in the context of a simple coupled equilibrium scheme. Studies carried out in this way provide quantitative insights into the degree to which a particular metal ion is capable of driving allosteric switching, and via ligand substitution, the extent to which individual coordination bonds establish structural linkage of allosteric metal and operator DNA-binding sites.

Allosteric coupling between transition metal-binding sites in homooligomeric metal sensor proteins.

Intracellular concentrations of transition metal ions are controlled at the transcriptional level by a panel of metalloregulatory proteins that collectively allow the cell to respond to changes in bioavailable metal concentration to elicit the appropriate cellular response, e.g., upregulation of genes coding for metal export or detoxification proteins in the event of metal excess. These proteins represent a specialized class of allosteric regulators that are ideal for studying ligand-mediated allostery in a comprehensive way due to the size, stability, reactivity, and the spectroscopic properties of transition metal ions as allosteric ligands. In addition to the commonly studied heterotropic regulation of metal binding and DNA binding, many of these proteins exhibit homotropic allostery, i.e., communication between two or more identical metal (ligand) binding sites on an oligomer. This chapter aims to guide the reader through the design and execution of experiments that allow quantification of the thermodynamic driving forces (ΔG (C), ΔH (C), and ΔS (C)) that govern both homotropic and heterotropic allosteric interactions in metal sensor proteins as well as the steps required to remove the influence of complex speciation from the measured parameter values.

The CRR1 nutritional copper sensor in Chlamydomonas contains two distinct metal-responsive domains.

Copper response regulator 1 (CRR1), an SBP-domain transcription factor, is a global regulator of nutritional copper signaling in Chlamydomonas reinhardtii and activates genes necessary during periods of copper deficiency. We localized Chlamydomonas CRR1 to the nucleus in mustard (Sinapis alba) seedlings, a location consistent with its function as a transcription factor. The Zn binding SBP domain of CRR1 binds copper ions in vitro. Cu(I) can replace Zn(II), but the Cu(II) form is unstable. The DNA binding activity is inhibited in vitro by Cu(II) or Hg(II) ions, which also prevent activation of transcription in vivo, but not by Co(II) or Ni(II), which have no effect in vivo. Copper inhibition of DNA binding is reduced by mutation of a conserved His residue. These results implicate the SBP domain in copper sensing. Deletion of a C-terminal metallothionein-like Cys-rich domain impacted neither nutritional copper signaling nor the effect of mercuric supplementation, but rendered CRR1 insensitive to hypoxia and to nickel supplementation, which normally activate the copper deficiency regulon in wild-type cells. Strains carrying the crr1-ΔCys allele upregulate ZRT genes and hyperaccumulate Zn(II), suggesting that the effect of nickel ions may be revealing a role for the C-terminal domain of CRR1 in zinc homeostasis in Chlamydomonas.

Application of isothermal titration calorimetry in bioinorganic chemistry.

The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC (p)°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (K (ITC), ΔH (ITC)). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal-buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.

Energetics of allosteric negative coupling in the zinc sensor S. aureus CzrA.

The linked equilibria of an allosterically regulated protein are defined by the structures, residue-specific dynamics and global energetics of interconversion among all relevant allosteric states. Here, we use isothermal titration calorimetry (ITC) to probe the global thermodynamics of allosteric negative regulation of the binding of the paradigm ArsR-family zinc sensing repressor Staphylococcus aureus CzrA to the czr DNA operator (CzrO) by Zn(2+). Zn(2+) binds to the two identical binding sites on the free CzrA homodimer in two discernible steps. A larger entropic driving force Delta(-TDeltaS) of -4.7 kcal mol(-1) and a more negative DeltaC(p) characterize the binding of the first Zn(2+) relative to the second. These features suggest a modest structural transition in forming the Zn(1) state followed by a quenching of the internal dynamics on filling the second zinc site, which collectively drive homotropic negative cooperativity of Zn(2+) binding (Delta(DeltaG) = 1.8 kcal mol(-1)). Negative homotropic cooperativity also characterizes Zn(2+) binding to the CzrA*CzrO complex (Delta(DeltaG) = 1.3 kcal mol(-1)), although the underlying energetics are vastly different, with homotropic Delta(DeltaH) and Delta(-TDeltaS) values both small and slightly positive. In short, Zn(2+) binding to the complex fails to induce a large structural or dynamical change in the CzrA bound to the operator. The strong heterotropic negative linkage in this system (DeltaG(c)(t) = 6.3 kcal mol(-1)) therefore derives from the vastly different structures of the apo-CzrA and CzrA*CzrO reference states (DeltaH(c)(t) = 9.4 kcal mol(-1)) in a way that is reinforced by a global rigidification of the allosterically inhibited Zn(2) state off the DNA (TDeltaS(c)(t) = -3.1 kcal mol(-1), i.e., DeltaS(c)(t) > 0). The implications of these findings for other metalloregulatory proteins are discussed.

Coronavirus N protein N-terminal domain (NTD) specifically binds the transcriptional regulatory sequence (TRS) and melts TRS-cTRS RNA duplexes.

All coronaviruses (CoVs), including the causative agent of severe acute respiratory syndrome (SARS), encode a nucleocapsid (N) protein that harbors two independent RNA binding domains of known structure, but poorly characterized RNA binding properties. We show here that the N-terminal domain (NTD) of N protein from mouse hepatitis virus (MHV), a virus most closely related to SARS-CoV, employs aromatic amino acid-nucleobase stacking interactions with a triple adenosine motif to mediate high-affinity binding to single-stranded RNAs containing the transcriptional regulatory sequence (TRS) or its complement (cTRS). Stoichiometric NTD fully unwinds a TRS-cTRS duplex that mimics a transiently formed transcription intermediate in viral subgenomic RNA synthesis. Mutation of the solvent-exposed Y127, positioned on the beta-platform surface of our 1.75 A structure, binds the TRS far less tightly and is severely crippled in its RNA unwinding activity. In contrast, the C-terminal domain (CTD) exhibits no RNA unwinding activity. Viruses harboring Y127A N mutation are strongly selected against and Y127A N does not support an accessory function in MHV replication. We propose that the helix melting activity of the coronavirus N protein NTD plays a critical accessory role in subgenomic RNA synthesis and other processes requiring RNA remodeling.

Thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the urease metallochaperone UreE.

The two Ni2+ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni2+ binding to the wild-type K. aerogenes UreE protein, and Ni2+ and Zn2+ binding to the wild-type UreE protein from Bacillus pasteurii. The stoichiometries and affinities obtained by ITC are in good agreement with previous equilibrium dialysis results, after differences in pH and buffer competition are considered, but the concentration of H144*UreE was found to have a significant effect on metal binding stoichiometry. While two metal ions bind to the H144*UreE dimer at concentrations <10 microM, three Ni2+ or Cu2+ ions bind to 25 microM dimeric protein with ITC data indicating sequential formation of Ni/Cu(H144*UreE)4 and then (Ni/Cu)2(H144*UreE)4, or Ni/Cu(H144*UreE)2, followed by the binding of four additional metal ions per tetramer, or two per dimer. The thermodynamics indicate that the latter two metal ions bind at sites corresponding to the two binding sites observed at lower protein concentrations. Ni2+ binding to UreE from K. aerogenes is an enthalpically favored process but an entropically driven process for the B. pasteurii protein, indicating chemically different Ni2+ coordination to the two proteins. A relatively small negative value of DeltaCp is associated with Ni2+ and Cu2+ binding to H144*UreE at low protein concentrations, consistent with binding to surface sites and small changes in the protein structure.

Metal-binding thermodynamics of the histidine-rich sequence from the metal-transport protein IRT1 of Arabidopsis thaliana.

The widespread ZIP family of transmembrane metal-transporting proteins is characterized by a large intracellular loop that contains a histidine-rich sequence whose biological role is unknown. To provide a chemical basis for this role, we prepared and studied a peptide corresponding to this sequence from the first iron-regulated transporter (IRT1) of Arabidopsis thaliana, which transports Fe2+ as well as Mn2+, Co2+, Zn2+, and Cd2+. Isothermal titration calorimetry (ITC) measurements, which required novel experiments and data analysis, and supporting spectroscopic methods were used to quantify IRT1's metal-binding affinity and associated thermodynamics. The peptide, PHGHGHGHGP, binds metal ions with 1:1 stoichiometry and stabilities that are consistent with the Irving-Williams series. Comparison of the metal-binding thermodynamics of the peptide with those of trien provides new insight about enthalpic and entropic contributions to the stability of the metal-peptide complex. Although Fe2+ and other IRT1-transported metal ions do not bind very tightly, this His-rich sequence has a very high entropy-driven affinity for Fe3+, which may have biological significance.