Zhiguang Xiao

Unification of the Copper(I) Binding Affinities of the Metallo-chaperones Atx1, Atox1, and Related Proteins DETECTION PROBES AND AFFINITY STANDARDS

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2]3− with formation constants β2 = 1017.2 and 1019.8 m−2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10−12 to 10−19 m. Dtt binds CuI with KD ∼10−15 m at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

Metal Binding Affinities of Arabidopsis Zinc and Copper Transporters: Selectivities Match the Relative, but Not the Absolute, Affinities of their Amino-Terminal Domains

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P(1B)-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn(2+), and the third transports Cu(+). Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu(I) binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn(2+)-ATPases. The three MBDs HMA2n (residues 1-79), HMA4n (residues 1-96), and HMA7n (residues 56-127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn(II) and Cu(I) of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn(II) are higher than that of HMA7n by a factor of 20-30, but the relative affinities for Cu(I) are inverted by a factor of 30-50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu(I) with an affinity between those of HMA4n and HMA7n but binds Zn(II) more weakly than either parent protein does. The four MBDs bind Cu(I) more strongly than Zn(II) by factors of >10(6). It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu(+) over Zn(2+). This information highlights the potential toxicity of Cu(+) in vivo and why copper sensor proteins are approximately 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein-protein interactions), and compartmentalization. The structure of Zn(II)-bound HMA4n defined by NMR confirmed the predicted ferredoxin betaalphabetabetaalphabeta fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed (113)Cd chemical shift in [(113)Cd]HMA4n was consistent with a Cd(II)S(2)OX (X = O or N) coordination sphere. The Zn(II) form of the Cu(I) transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn(II)-HMA7n)(m)]. Each Zn(II) ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.

A C-terminal domain of the membrane copper pump Ctr1 exchanges copper(I) with the copper chaperone Atx1.

A cloned C-terminal domain of the yeast high-affinity copper uptake pump Ctr1 exchanges Cu(I) rapidly with the yeast copper chaperone Atx1: 10(-2) < Kex < 10(+2).

The challenges of using a copper fluorescent sensor (CS1) to track intracellular distributions of copper in neuronal and glial cells

Copper is an essential biometal involved in critical cell functions including respiration. However, the mechanisms controlling its sub-cellular localization during health and disease remain poorly understood. This is partially due to the difficulty of detecting a metal ion that is bound tightly to metallo-chaperone and detoxification molecules in the cell. A BODIPY-based Cu fluorescent probe CS1 (Cu sensor 1) has been applied in innovative attempts to visualize monovalent Cu pools within cells (Zeng et al., J. Am. Chem. Soc., 2006, 128, 10–11). Inspired by this work, we sought to use CS1 to identify sub-cellular localization of Cu delivered to M17 neuronal or U87MG glial cells by a cell-permeable bis(thiosemicarbazonato)Cu(II) complex, CuII(gtsm). This complex increases cellular Cu concentrations by factors of 10–100 when compared to treatment with equivalent concentrations of CuCl2 (Donnelly et al., J. Biol. Chem., 2008, 283, 4568–4577). However, we were unable to identify any specific increase in CS1 fluorescence in neurons or glia treated with CuCl2 or with CuII(gtsm), despite controls revealing a large increase in total cellular Cu with the latter treatment. Further in vitro characterization of CS1 suggests that, consistent with its relatively weak affinity for CuI (KD ≈ 10−11 M), it is unlikely to compete with endogenous proteins with sub-picomolar affinities, nor with glutathione, the endogenous redox buffer essential for functional maintenance of many proteins, including those that bind CuI. Moreover, we show that CS1 is localized predominantly to lysosomes and that the observed background fluorescence may be attributed to increased concentrations of apo-CS1 in this organelle or to the probe gaining access to CuI made available via recycling of nutrient Cu in the acidic lysosome. It was possible to observe a consistent increase in CS1 fluorescence in neuronal cells exposed to stress. For example, treatment with buthionine sulfoximine decreased cellular glutathione levels and led to enhanced CS1 fluorescence, but the total cellular Cu levels did not correlate with the increased fluorescence. In addition, cells treated with reagents that are known to alter cellular pH homeostasis provided an enhanced fluorescence. Our findings demonstrate that the source of enhanced CS1 fluorescence in Cu-supplemented cells must be interpreted with caution. It may be a consequence of altered cell pH, compromised vesicle maturation, increased CS1 uptake and/or trapping of CS1 in the lysosomal compartment.

Unprecedented binding cooperativity between Cu(I) and Cu(II) in the copper resistance protein CopK from Cupriavidus metallidurans CH34: implications from structural studies by NMR spectroscopy and X-ray crystallography.

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. This ability arises primarily from the presence of a large plasmid pMOL30 which includes a cluster of 19 cop genes that respond to copper. One of the protein products CopK is induced at high levels and is expressed to the periplasm as a small soluble protein (8.3 kDa). Apo-CopK associates in solution to form a dimer (K(D) approximately 10(-5) M) whose structure was defined by NMR and X-ray crystallography. The individual molecules feature two antiparallel beta-sheets arranged in a sandwich-like structure and interact through C-terminal beta-strands. It binds Cu(II) with low affinity (K(D)(Cu(II)) > 10(-6) M) but Cu(I) with high affinity (K(D)(Cu(I)) = 2 x 10(-11) M). Cu(I)-CopK was also a dimer in the solid state and featured a distorted tetrahedral site Cu(I)(S-Met)(3)(NCS). The isothiocyanato ligand originated from the crystallization solution. Binding of Cu(I) or Ag(I), but not of Cu(II), favored the monomeric form in solution. While Ag(I)-CopK was stable as isolated, Cu(I)-CopK was moderately air-sensitive due to a strong binding cooperativity between Cu(I) and Cu(II). This was documented by determination of the Cu(I) and Cu(II) binding affinities in the presence of the other ion: K(D)(Cu(I)) = 2 x 10(-13) M and K(D)(Cu(II)) = 3 x 10(-12) M, that is, binding of Cu(II) increased the affinity for Cu(I) by a factor of approximately 10(2) and binding of Cu(I) increased the affinity for Cu(II) by a factor of at least 10(6). Stable forms of both Cu(I)Cu(II)-CopK and Ag(I)Cu(II)-CopK were isolated readily. Consistent with this unprecedented copper binding chemistry, NMR spectroscopy detected three distinct forms: apo-CopK, Cu(I)-CopK and Cu(I)Cu(II)-CopK that do not exchange on the NMR time scale. This information provides a valuable guide to the role of CopK in copper resistance.

Copper Resistance in E. coli: The Multicopper Oxidase PcoA Catalyzes Oxidation of Copper(I) in CuICuII‐PcoC

es that three soluble proteins (PcoA, PcoC, PcoE in E. coli) are expressed to the periplasm, that two copper pumps (PcoB, PcoD) are present in the outer and inner membranes, and that there is a copper sensing system (PcoS, PcoR). PcoC and CopC are highly homologous b-barrels (~11 kDa) that bind both Cu and Cu at sites separated by about 30 6. These sites are tailored to bind their individual ions with high affinity (KD~10 m): CuACHTUNGTRENNUNG(His) ACHTUNGTRENNUNG(Met)2or3 (trigonal or tetrahedral), Cu ACHTUNGTRENNUNG(His)2ACHTUNGTRENNUNG(N-term) ACHTUNGTRENNUNG(OH2) (square planar). [5, 6] Each shows little affinity for the other ion. All possible types of intermolecular copper transfer reactions have been demonstrated: oxidative transfer from the Cu site to the Cu site, reductive transfer from the Cu site to the Cu site, and non-redox transfers between Cu and Cu sites. This versatile chemistry is consistent with a role for PcoC as a copper carrier (chaperone) in the oxidizing periplasm, but which functions are employed remains unknown. apo-PcoA (63.9 kDa) was over-expressed in E. coli and isolated in high purity in the presence of ethylenediaminetetraacetate (EDTA), dithiothreitol (DTT), and glycerol (Figure S1). The protein ran as a monomer on a Superdex-75 size exclusion column (Figure S1). Incubation with more than five equiv of Cuaq in the presence of glutathione and removal of unbound ions by gel filtration led to optimal development of an absorbance spectrum characteristic of a multicopper oxidase (type 1 center : 600 nm (e, 4000m 1 cm ) ; OH-bridged type 3 binuclear center : ~340 nm sh; A280/A600, 23.0; Figure S2). Both type 1 and type 2 Cu centers were also detected in the frozen EPR spectrum (type 1 center: g?=2.07, gk =2.28, Ak =8.5G 10 3 cm ; type 2 center: g?=2.07, gk=2.30, Ak =17G 10 3 cm ; Figure S3). Isolated holo-PcoA contained 4.3 (0.4s) equiv of copper and exhibited phenol oxidase activity with substrate 2,6-dimethoxyphenol (DMP) at pH 7 (Figure S4; 550 mm DMP per mm PcoA per min), a feature of most multicopper oxidases. Unexpectedly, this enzyme exhibited maximal activity in the absence of added Cuaq, whereas related multicopper oxidases require this ion in excess to induce maximal phenol oxidase activity. Reaction of holo-PcoA with air-stable CuCu-PcoC under catalytic conditions (1:50) in air-saturated MOPS buffer (pH 7) led to quantitative generation of product Cu-PcoC (represented as C Figure 1); this is consistent with oxidation of bound Cu. The catalysis was suppressed dramatically in deoxygenated buffer (Figure S5), consistent with O2 acting as the oxidant. The product protein &Cu II has little affinity for co-product Cuaq, which appeared to be ACHTUNGTRENNUNGreleased into solution. Clean regeneration of CuCu-PcoC occurred upon addition of reductant NH2OH (that is, Figure 1D converted to Figure 1A). This is a robust catalyst : there was negligible loss of activity after four cycles of oxidation and ACHTUNGTRENNUNGreduction. The product PcoC protein &Cu suppressed cuprous oxidase activity, consistent with a previous suggestion that PcoC interacts with PcoA. Inclusion of increasing concentrations of &Cu in the initial reaction mixture in Figure 1 led to proportional decreases in the rate of the catalytic reaction, whereas addition of generic proteins such as lysozyme had no effect (Figure 2). These results, when coupled with the high affinity of PcoC for Cu (KD~10 m), demonstrate that PcoA catalyzes the oxidation of Cu bound in CuCu-PcoC. They suggest that PcoA and PcoC cooperate to convert Cu into the less toxic Cu in the O2-rich periplasm. This system exhibits two new features: cuprous oxidase ACHTUNGTRENNUNGactivity with a likely biological partner and maximal phenol ACHTUNGTRENNUNGoxidase activity in the absence of excess Cuaq. In seeking a model for this behavior, it was noted that PcoA has some homology (23%) with the tolerance enzyme CueO and, impor[a] K. Y. Djoko, Dr. Z. Xiao, Prof. A. G. Wedd School of Chemistry and Bio21 Research Institute, University of Melbourne Parkville, Victoria 3010 (Australia) Fax: (+61)3-9347-5180 E-mail : z.xiao@unimelb.edu.au

Mutation of the surface valine residues 8 and 44 in the rubredoxin from Clostridium pasteurianum : solvent access versus structural changes as determinants of reversible potential

Abstract The Pri sidechains of two adjacent valine residues, V8 and V44, define the surface of the rubredoxin from Clostridium pasteurianum and control access to its Fe(S-Cys)4 active site. To assess the effect of systematic change of the steric bulk of the alkyl sidechains, eight single and three double mutant proteins have been isolated which vary G (H), A (Me), V (Pri), L (Bui) and I (Bus) at those positions. X-ray crystal structures of the FeIII forms of the V44A and V44I proteins are reported. Positive shifts in reversible potential of up to 116 mV are observed and attributed to increased polarity around the Fe(S-Cys)4 site induced by (1) changes in protein backbone conformation driven by variation of the steric demands of the sidechain substituents and (2) changes in solvent access to the sidechains of ligands C9 and C42. Data for the V44A mutant show that a minor change in the steric requirements of a surface residue can introduce a NH···Sγ hydrogen bond at the active site and lead to a shift in potentialof +50 mV.

Solvation Effects on S K-Edge XAS Spectra of Fe−S Proteins: Normal and Inverse Effects on WT and Mutant Rubredoxin

S K-edge X-ray absorption spectroscopy (XAS) was performed on wild type Cp rubredoxin and its Cys --> Ser mutants in both solution and lyophilized forms. For wild type rubredoxin and for the mutants where an interior cysteine residue (C6 or C39) is substituted by serine, a normal solvent effect is observed, that is, the S covalency increases upon lyophilization. For the mutants where a solvent accessible surface cysteine residue is substituted by serine, the S covalency decreases upon lyophilization which is an inverse solvent effect. Density functional theory (DFT) calculations reproduce these experimental results and show that the normal solvent effect reflects the covalency decrease due to solvent H-bonding to the surface thiolates and that the inverse solvent effect results from the covalency compensation from the interior thiolates. With respect to the Cys --> Ser substitution, the S covalency decreases. Calculations indicate that the stronger bonding interaction of the alkoxide with the Fe relative to that of thiolate increases the energy of the Fe d orbitals and reduces their bonding interaction with the remaining cysteines. The solvent effects support a surface solvent tuning contribution to electron transfer, and the Cys --> Ser result provides an explanation for the change in properties of related iron-sulfur sites with this mutation.

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.

Protonless 13C direct detection NMR: Characterization of the 37 kDa trimeric protein CutA1

The major limitation of nuclear magnetic resonance spectroscopy arises from the increase of nuclear transverse relaxation rates with increasing molecular mass. This causes reduction in spectral resolution and coherence transfer efficiency. The use of 2H‐labeling to eliminate 1H‐mediated relaxation pathways and the constructive use of cross correlation effects (TROSY, CRINEPT) alleviate the phenomenon. An alternative approach is to use direct detection of heteronuclei. Specifically designed 13C direct detection experiments can complement the set of 1H‐based NMR experiments commonly used for structure determination providing an additional source of information less affected by the detrimental transverse relaxation effect. We applied this novel methodology to the study of the CutA1 protein (12.3 kDa) from E. coli that forms a homotrimer in solution with a total molecular mass of 37 kDa. In this work we demonstrate that the information available from 13C direct detection experiments makes it possible to completely assign the NMR resonances of the backbone of this 37 kDa trimeric protein without the need of deuteration. The structural and dynamical knowledge obtained for this system may contribute to understand its biological role. Proteins 2008.