Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B12.

The acquisition of CoII by the corrin component of vitamin B12 follows one of two distinct pathways, referred to as early or late CoII insertion. The late insertion pathway exploits a CoII metallochaperone (CobW) from the COG0523 family of G3E GTPases, while the early insertion pathway does not. This provides an opportunity to contrast the thermodynamics of metalation in a metallochaperone-requiring and a metallochaperone-independent pathway. In the metallochaperone-independent route, sirohydrochlorin (SHC) associates with the CbiK chelatase to form CoII-SHC. CoII-buffered enzymatic assays indicate that SHC binding enhances the thermodynamic gradient for CoII transfer from the cytosol to CbiK. In the metallochaperone-dependent pathway, hydrogenobyrinic acid a,c-diamide (HBAD) associates with the CobNST chelatase to form CoII-HBAD. Here, CoII-buffered enzymatic assays indicate that CoII transfer from the cytosol to HBAD-CobNST must somehow traverse a highly unfavorable thermodynamic gradient for CoII binding. Notably, there is a favorable gradient for CoII transfer from the cytosol to the MgIIGTP-CobW metallochaperone, but further transfer of CoII from the GTP-bound metallochaperone to the HBAD-CobNST chelatase complex is thermodynamically unfavorable. However, after nucleotide hydrolysis, CoII transfer from the chaperone to the chelatase complex is calculated to become favorable. These data reveal that the CobW metallochaperone can overcome an unfavorable thermodynamic gradient for CoII transfer from the cytosol to the chelatase by coupling this process to GTP hydrolysis.

Metalation calculators for E. coli strain JM109 (DE3): aerobic, anaerobic, and hydrogen peroxide exposed cells cultured in LB media.

Three Web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of Escherichia coli that is commonly used in molecular biology and biochemistry research, e.g. in the production of recombinant proteins. Metal availabilities have been examined for cells grown in Luria-Bertani (LB) medium aerobically, anaerobically, and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by quantitative polymerase chain reaction (qPCR). The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterized in related bacterial cells enabling gene expression to be read out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial that discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.

Protein metalation in biology.

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.

Insights into the Antibacterial Mechanism of Action of Chelating Agents by Selective Deprivation of Iron, Manganese, and Zinc.

Bacterial growth and proliferation can be restricted by limiting the availability of metal ions in their environment. Humans sequester iron, manganese, and zinc to help prevent infection by pathogens, a system termed nutritional immunity. Commercially used chelants have high binding affinities with a variety of metal ions, which may lead to antibacterial properties that mimic these innate immune processes. However, the modes of action of many of these chelating agents in bacterial growth inhibition and their selectivity in metal deprivation in cellulo remain ill-defined. We address this shortcoming by examining the effect of 11 chelators on Escherichia coli growth and their impact on the cellular concentration of five metals. The following four distinct effects were uncovered: (i) no apparent alteration in metal composition, (ii) depletion of manganese alongside reductions in iron and zinc levels, (iii) reduced zinc levels with a modest reduction in manganese, and (iv) reduced iron levels coupled with elevated manganese. These effects do not correlate with the absolute known chelant metal ion affinities in solution; however, for at least five chelators for which key data are available, they can be explained by differences in the relative affinity of chelants for each metal ion. The results reveal significant insights into the mechanism of growth inhibition by chelants, highlighting their potential as antibacterials and as tools to probe how bacteria tolerate selective metal deprivation. IMPORTANCE Chelating agents are widely used in industry and consumer goods to control metal availability, with bacterial growth restriction as a secondary benefit for preservation. However, the antibacterial mechanism of action of chelants is largely unknown, particularly with respect to the impact on cellular metal concentrations. The work presented here uncovers distinct metal starvation effects imposed by different chelants on the model Gram-negative bacterium Escherichia coli. The chelators were studied both individually and in pairs, with the majority producing synergistic effects in combinations that maximize antibacterial hostility. The judicious selection of chelants based on contrasting cellular effects should enable reductions in the quantities of chelant required in numerous commercial products and presents opportunities to replace problematic chemistries with biodegradable alternatives.

Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site.

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed "methionine track" leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

Principles and practice of determining metal-protein affinities.

Metal ions play many critical roles in biology, as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal-protein affinity, expressed conveniently by the metal dissociation constant, KD, describes the thermodynamic strength of a metal-protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal-protein affinities. We review a selection of spectroscopic probes which can be used to determine protein affinities for essential biological transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent experimental design can be applied to determine accurate KD values.

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII.

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

The requirement for cobalt in vitamin B12: A paradigm for protein metalation.

Vitamin B12, cobalamin, is a cobalt-containing ring-contracted modified tetrapyrrole that represents one of the most complex small molecules made by nature. In prokaryotes it is utilised as a cofactor, coenzyme, light sensor and gene regulator yet has a restricted role in assisting only two enzymes within specific eukaryotes including mammals. This deployment disparity is reflected in another unique attribute of vitamin B12 in that its biosynthesis is limited to only certain prokaryotes, with synthesisers pivotal in establishing mutualistic microbial communities. The core component of cobalamin is the corrin macrocycle that acts as the main ligand for the cobalt. Within this review we investigate why cobalt is paired specifically with the corrin ring, how cobalt is inserted during the biosynthetic process, how cobalt is made available within the cell and explore the cellular control of cobalt and cobalamin levels. The partitioning of cobalt for cobalamin biosynthesis exemplifies how cells assist metalation.

The Human Amyloid Precursor Protein Binds Copper Ions Dominated by a Picomolar-Affinity Site in the Helix-Rich E2 Domain.

A manifestation of Alzheimer's disease (AD) is the aggregation in the brain of amyloid ß (Aß) peptides derived from the amyloid precursor protein (APP). APP has been linked to modulation of normal copper homeostasis, while dysregulation of Aß production and clearance has been associated with disruption of copper balance. However, quantitative copper chemistry on APP is lacking, in contrast to the plethora of copper chemistry available for Aß peptides. The soluble extracellular protein domain sAPPα (molar mass including post-translational modifications of ∼100 kDa) has now been isolated in good yield and high quality. It is known to feature several copper binding sites with different affinities. However, under Cu-limiting conditions, it binds either Cu(I) or Cu(II) with picomolar affinity at a single site (labeled M1) that is located within the APP E2 subdomain. M1 in E2 was identified previously by X-ray crystallography as a Cu(II) site that features four histidine side chains (H313, H386, H432, and H436) as ligands. The presence of CuII(His)4 is confirmed in solution at pH ≤7.4, while Cu(I) binding involves either the same ligands or a subset. The binding affinities are pH-dependent, and the picomolar affinities for both Cu(I) and Cu(II) at pH 7.4 indicate that either oxidation state may be accessible under physiological conditions. Redox activity was observed in the presence of an electron donor (ascorbate) and acceptor (dioxygen). A critical analysis of the potential biological implications of these findings is presented.

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

Copper binding and redox chemistry of the Aß16 peptide and its variants: insights into determinants of copper-dependent reactivity.

The metal-binding sites of Aß peptides are dictated primarily by the coordination preferences of the metal ion. Consequently, Cu(i) is typically bound with two His ligands in a linear mode while Cu(ii) forms a pseudo-square planar stereochemistry with the N-terminal amine nitrogen acting as an anchoring ligand. Several distinct combinations of other groups can act as co-ligands for Cu(ii). A population of multiple binding modes is possible with the equilibrium position shifting sensitively with solution pH and the nature of the residues in the N-terminal region. This work examined the Cu(ii) chemistry of the Aß16 peptide and several variants that targeted these binding modes. The results are consistent with: (i) at pH < 7.8, the square planar site in CuII-Aß16 consists primarily of a bidentate ligand provided by the carboxylate sidechain of Asp1 and the N-terminal amine supported by the imidazole sidechains of two His residues (designated here as component IA); it is in equilibrium with a less stable component IB in which the carboxylate ligand is substituted by the Asp1-Ala2 carbonyl oxygen. (ii) Both IA and IB convert to a common component II (apparent transition pKa ∼7.8 for IA and ∼6.5 for IB, respectively) featuring a tridentate ligand consisting of the N-terminal amine, the Asp1-Ala2 amide and the Ala2-Pro3 carbonyl; this stereochemistry is stabilized by two five-membered chelate rings. (iii) Component IA is stabilized for variant Aß16-D1H, components I (both IA and IB) are imposed on Aß16-A2P while the less stable IB is enforced on Aß16-D1A (which is converted to component II at pH ∼6.5); (iv) components IA and IB share two His ligands with Cu(i) and are more reactive in redox catalysis than component II that features a highly covalent and less reactive amide N- ligand. The redox activity of IA is further enhanced for peptides with a His1 N-terminus that may act as a ligand for either Cu(i) or Cu(ii) with lower re-organization energy required for redox-shuttling. This study provided insights into the determinants that regulate the reactivity of Cu-Aß complexes.

A set of robust fluorescent peptide probes for quantification of Cu(ii) binding affinities in the micromolar to femtomolar range.

Reliable quantification of copper binding affinities and identification of the binding sites provide a molecular basis for an understanding of the nutritional roles and toxic effects of copper ions. Sets of chromophoric probes are now available that can quantify Cu(i) binding affinities from nanomolar to attomolar concentrations on a unified scale under in vitro conditions. Equivalent probes for Cu(ii) are lacking. This work reports development of a set of four fluorescent dansyl peptide probes (DP1-4) that can quantify Cu(ii) binding affinities from micromolar to femtomolar concentrations, also on a unified scale. The probes were constructed by conjugation of a dansyl group to four short peptides of specific design. Each was characterised by its dissociation constant KD, its pH dependence and the nature of its binding site. One equivalent of Cu(ii) is bound by the individual probes that display different and well-separated affinities at pH 7.4 (log KD = -8.1, -10.1, -12.3 and -14.1, respectively). Intense fluorescence is emitted at λmax ∼ 550 nm upon excitation at ∼330 nm. Binding of Cu(ii) quenches the fluorescence intensity linearly until one equivalent of Cu(ii) is bound. Multiple approaches and multiple affinity standards were employed to ensure reliability. Selected examples of application to well-characterised Cu(ii) binding peptides and proteins are presented. These include Aß16 peptides, two naturally occurring Cu(ii)-chelating motifs in human serum and cerebrospinal fluid with sequences GHK and DAHK and two copper binding proteins, CopC from Pseudomonas syringae and PcoC from Escherichia coli. Previously reported affinities are reproduced, demonstrating that peptides DP1-4 form a set of robust and reliable probes for Cu(ii) binding to peptides and protein targets.

CopC protein from Pseudomonas fluorescens SBW25 features a conserved novel high-affinity Cu(II) binding site.

Copper homeostasis in the bacterium Pseudomonas fluorescens SBW25 appears to be mediated mainly via chromosomal cue and cop systems. Under elevated copper levels that induce stress, the cue system is activated for expression of a P1B-type ATPase to remove excess copper from the cytosol. Under copper-limiting conditions, the cop system is activated to express two copper uptake proteins, Pf-CopCD, to import this essential nutrient. Pf-CopC is a periplasmic copper chaperone that may donate copper to the inner membrane transporter Pf-CopD for active copper importation. A database search revealed that Pf-CopC belongs to a new family of CopC proteins (designated Type B in this work) that differs significantly from the known CopC proteins of Type A that possess two separated binding sites specific for Cu(I) and Cu(II). This article reports the isolation and characterization of Pf-CopC and demonstrates that it lacks a Cu(I) binding site and possesses a novel Cu(II) site that binds Cu(II) with 100 times stronger affinity than do the Type A proteins. Presumably, this is a requirement for a copper uptake role under copper-limiting conditions. The Cu(II) site incorporates a highly conserved amino terminal copper and nickel (ATCUN) binding motif, NH2-Xxx-Xxx-His, but the anticipated ATCUN binding mode is prevented by a thermodynamically more favorable binding mode comprising His1 as a key bidentate ligand and His3 and His85 as co-ligands. However, upon His1 mutation, the ATCUN binding mode is adopted. This work demonstrates how a copper chaperone may fine tune its copper binding site to meet new challenges to its function.

An integrated study of the affinities of the Aß16 peptide for Cu(I) and Cu(II): implications for the catalytic production of reactive oxygen species.

A new fluorescent probe Aß16wwa based upon the Aß16 peptide has been developed with two orders of magnitude greater fluorescence intensity for sensitive detection of interactions with Cu(II). In combination with the Cu(I) probe Ferene S, it is confirmed that the Aß16 peptide binds either Cu(I) or Cu(II) with comparable affinities at pH 7.4 (log K = -10.4; log K = -10.0). It follows from this property that the Cu-Aß16 complex is a robust if slow catalyst for the aerial oxidation of ascorbate with H2O2 as primary product (initial rate, ∼0.63 min(-1) for Cu-Aß16 versus >2.5 min(-1) for Cuaq(2+)). An integrated study of variants of this peptide identifies the major ligands and binding modes involved in its copper complexes in solution. The dependence of K upon pH is consistent with a two-coordinate Cu(I) site in which dynamic processes exchange Cu(I) between the three available pairs of imidazole sidechains provided by His6, His13 and His14. The N-terminal amine is not involved in Cu(I) binding but is a key ligand for Cu(II). Acetylation of the N-terminus alters the redox thermodynamic gradient for the Cu centre and suppresses its catalytic activity considerably. The data indicate the presence of dynamic processes that exchange Cu(II) between the three His ligands and backbone amide at physiological pH. His6 is identified as a key ligand for catalysis as its presence minimises the pre-organisation energy required for interchange of the two copper redox sites. These new thermodynamic data strengthen structural interpretations for the Cu-Aß complexes and provide valuable insights into the molecular mechanism by which copper chemistry may induce oxidative stress in Alzheimer's disease.

Quantification of copper binding to amyloid precursor protein domain 2 and its Caenorhabditis elegans ortholog. Implications for biological function.

Aberrant regulation of transition metals and the resultant disregulation of neuronal reactive oxygen species (ROS) are considered significant in the etiology of Alzheimer's disease (AD). We determined the solution structure of the D2 domain of APL-1 (APL1-D2), the Caenorhabditis elegans ortholog of the amyloid precursor protein domain 2 (APP-D2). The copper binding affinities of APL1-D2 and APP-D2 were estimated and the ability of their copper complexes to promote formation of ROS was tested. The two protein domains are isostructural, consisting of a three-stranded ß-sheet packed against a short α-helix in a ßαßß fold. A six-residue insert in APL1-D2, absent in APP-D2, forms an extended loop. The putative copper binding ligands in APP-D2 are not conserved in APL1-D2 and this delineates a clear difference between them. APL1-D2 and APP-D2 bind one equivalent of Cu(I) weakly, with dissociation constants KD ∼10(-8.6) M and ~10(-10) M, respectively, and up to two equivalents of Cu(II) with KD values in the range 10(-6) -10(-8) M. The relative abilities of APL1-D2, APP-D2 and amyloid-ß (Aß) copper complexes to generate H2O2 correspond to their copper binding affinities. Copper affinities for Aß (KD ~ 10(-10) M for both Cu(I) and Cu(II)) and APP-D2 are in a range allowing redox cycling to occur, albeit less efficiently for APP-D2. However, APL1-D2 binds Cu(I) and Cu(II) too weakly to sustain catalysis which further suggests that it plays no significant role in copper handling in C. elegans. Overall, the data are consistent with a possible role in copper homeostasis for APP-D2, but not APL1-D2.