Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.     

Probing determinants of the metal ion selectivity in carbonic anhydrase using mutagenesis

Few studies measuring thermodynamic metal ion selectivity of metalloproteins have been performed, and the major determinants of metal ion selectivity in proteins are not yet well understood. Several features of metal ion binding sites and metal coordination have been hypothesized to alter the transition metal selectivity of chelators, including (1) the polarizability of the coordinating atom, (2) the relative sizes of the binding site and the metal ion, and (3) the metal ion binding site geometry. To test these hypotheses, we have measured the metal ion affinity and selectivity of a prototypical zinc enzyme, human carbonic anhydrase II (CAII), and a number of active site variants where one of the coordinating ligands is substituted by another side chain capable of coordinating metal. CAII and almost all of the variants follow the inherent metal ion affinity trend suggested by the Irving-Williams series, demonstrating that this trend operates within proteins as well as within small molecule chelators and may be a dominant factor in metal ion selectivity in biology. Neither the polarizability of the liganding side chains nor the size of the metal ion binding site correlates strongly with metal ion specificity; instead, changes in metal ion specificity in the variants correlate with the preferred coordination number and geometry of the metal ion. This correlation suggests that a primary feature driving deviations from the inherent ligand affinity trend is the positioning of active site groups such that a given metal ion can adopt a preferred coordination number/geometry.     

Effect of replacement of "zinc finger" zinc on estrogen receptor DNA interactions.

Exposure of bovine estrogen receptor to the metal chelators EDTA and 1,10-phenanthroline results in a loss of nonspecific DNA binding, presumably because of the removal of "zinc finger" zinc. Nonspecific DNA binding, as measured by a DNA-cellulose binding assay, can be restored by dialysis of the aporeceptor against buffer containing zinc, cadmium, and cobalt but not with buffer containing copper or nickel. More detailed studies were carried out using a bacterially expressed polypeptide encompassing the DNA binding domain of the human estrogen receptor. Apopolypeptide fails to bind DNA specifically, as measured by mobility shift assay using a consensus estrogen response element hexamer containing oligonucleotide, but DNA binding was restored by dialysis of the apopolypeptide against buffer containing zinc, cadmium, and cobalt but not with buffer containing copper or nickel. Dissociation constants of zinc- and cadmium-reconstituted polypeptide for the estrogen response element hexamer (66 and 48 nM, respectively) are virtually indistinguishable from native polypeptide (Kd = 48 nM) whereas cobalt-reconstituted polypeptide has a lower affinity (Kd = 720 nM). However, native, zinc-, cadmium-, and cobalt-reconstituted polypeptides gave identical results in a methylation interference assay. Competition experiments with zinc and copper or nickel suggest that copper and nickel are able to bind to zinc finger residues but do so nonproductively. The relative affinities copper greater than cadmium greater than zinc greater than cobalt greater than nickel for the polypeptide were determined by a zinc blot competition assay. The ability of cadmium and cobalt to substitute for zinc in the zinc fingers demonstrates a structural "flexibility" in the DNA binding domain as each of these metals has slightly different ionic radii. On the other hand, subtle differences in DNA binding affinity and/or specificity could exist, which may not be detectable here. Also, the ability of metals to substitute for zinc in the DNA binding domain suggests that metal substitution in these zinc fingers in vivo may be of relevance to the toxicity and/or carcinogenicity of some of these metals.     

Deciphering metal ion preference and primary coordination sphere robustness of a designed zinc finger with high‐resolution mass spectrometry

Small zinc finger (ZnF) motifs are promising molecular scaffolds for protein design owing to their structural robustness and versatility. Moreover, their characterization provides important insights into protein folding in general. ZnF motifs usually possess an exceptional specificity and high affinity towards Zn(II) ion to drive folding. While the Zn(II) ion is canonically coordinated by two cysteine and two histidine residues, many other coordination spheres also exist in small ZnFs, all having four amino acid ligands. Here we used high‐resolution mass spectrometry to study metal ion binding specificity and primary coordination sphere robustness of a designed zinc finger, named MM1. Based on the results, MM1 possesses high specificity for zinc with sub‐micromolar binding affinity. Surprisingly, MM1 retains metal ion binding affinity even in the presence of selective alanine mutations of the primary zinc coordinating amino acid residues.     

Zinc-mediated amino acid discrimination in cysteinyl-tRNA synthetase

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves a high level of amino acid specificity without an editing reaction. The crystal structure of CysRS bound to substrate cysteine suggested that direct thiol coordination to a tightly bound zinc ion at the base of the active site is the primary determinant of selectivity against non-cognate amino acids. This hypothesis has now been supported by spectroscopic studies of cobalt-substituted CysRS. Binding of cysteine, but not non-cognate amino acids, induces high absorption in the ligand-to-metal charge transfer region, providing evidence for formation of a metal-thiolate bond. In addition, mutations in the zinc ligands alter the absorption spectrum without reducing the discrimination against non-cognate amino acids. These results argue strongly for a major role for the zinc ion in amino acid discrimination by CysRS, where the tight zinc-thiolate interaction and the strict structural geometry of the metal ion are sufficient to reject serine by more than 20,000-fold at the binding step.     

Prediction of structures of zinc‐binding proteins through explicit modeling of metal coordination geometry

Metal ions play an essential role in stabilizing protein structures and contributing to protein function. Ions such as zinc have well‐defined coordination geometries, but it has not been easy to take advantage of this knowledge in protein structure prediction efforts. Here, we present a computational method to predict structures of zinc‐binding proteins given knowledge of the positions of zinc‐coordinating residues in the amino acid sequence. The method takes advantage of the “atom‐tree” representation of molecular systems and modular architecture of the Rosetta3 software suite to incorporate explicit metal ion coordination geometry into previously developed de novo prediction and loop modeling protocols. Zinc cofactors are tethered to their interacting residues based on coordination geometries observed in natural zinc‐binding proteins. The incorporation of explicit zinc atoms and their coordination geometry in both de novo structure prediction and loop modeling significantly improves sampling near the native conformation. The method can be readily extended to predict protein structures bound to other metal and/or small chemical cofactors with well‐defined coordination or ligation geometry.     

Zinc coordination geometry and ligand binding affinity: the structural and kinetic analysis of the second-shell serine 228 residue and the methionine 180 residue of the aminopeptidase from Vibrio proteolyticus

The chemical properties of zinc make it an ideal metal to study the role of coordination strain in enzymatic rate enhancement. The zinc ion and the protein residues that are bound directly to the zinc ion represent a functional charge/dipole complex, and polarization of this complex, which translates to coordination distortion, may tune electrophilicity, and hence, reactivity. Conserved protein residues outside of the charge/dipole complex, such as second-shell residues, may play a role in supporting the electronic strain produced as a consequence of functional polarization. To test the correlation between charge/dipole polarity and ligand binding affinity, structure-function studies were carried out on the dizinc aminopeptidase from Vibrio proteolyticus. Alanine substitutions of S228 and M180 resulted in catalytically diminished enzymes whose crystal structures show very little change in the positions of the metal ions and the protein residues. However, more detailed inspections of the crystal structures show small positional changes that account for differences in the zinc ion coordination geometry. Measurements of the binding affinity of leucine phosphonic acid, a transition state analogue, and leucine, a product, show a correlation between coordination geometry and ligand binding affinity. These results suggest that the coordination number and polarity may tune the electrophilicity of zinc. This may have provided the evolving enzyme with the ability to discriminate between reaction coordinate species.     

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(I) per CopZ and two copper(I) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(I)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(I)2CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(I)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper, from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(I) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange; a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.     

Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA

Recent studies on metalloregulatory proteins suggest that coordination number/geometry and metal ion availability in a host cytosol are key determinants for biological specificity. Here, we investigate the contribution that individual metal ligands of the alpha5 sensing site of Staphylococcus aureus CzrA (Asp84, His86, His97', and His100') make to in vitro metal ion binding affinity, coordination geometry, and allosteric negative regulation of DNA operator/promoter region binding. All ligand substitution mutants exhibit significantly reduced metal ion binding affinity (K(Me)) by > or =10(3) M(-1). Substitutions of Asp84 and His97 give rise to non-native coordination geometries upon metal binding and are non-functional in allosteric coupling of metal and DNA binding (DeltaG(coupling) approximately 0 kcal mol(-1)). In contrast, His86 and His100 could be readily substituted with potentially liganding (Asp, Glu) and poorly liganding (Asn, Gln) residues with significant native-like tetrahedral metal coordination geometry retained in these mutants, leading to strong functional coupling (DeltaG(coupling) > or = +3.0 kcal mol(-1)). 1H-(15)N heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4 degrees structural switching on binding Zn(II), while D84N, H97N and H97D CzrAs do not. Thus, only those variant CzrAs that retain some tetrahedral coordination geometry characteristic of wild-type CzrA upon metal binding are capable of driving 4 degrees structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of K(Me).     

Localization of the Cl-/HCO3- anion exchanger binding site to the amino-terminal region of carbonic anhydrase II

Human carbonic anhydrase II (CAII) possesses a binding site for an acidic motif (D887ADD) within the carboxyl-terminal region (Ct) of the human erythrocyte chloride/bicarbonate anion exchanger, AE1. In this study, the amino acid sequence comprising this AE1 binding site was localized to the first 17 residues of CAII, which form a basic patch on the surface of the protein. Truncation of the amino terminal of CAII by five residues resulted in a 3-fold reduction in the apparent affinity of the interaction with a GST fusion protein of the Ct of AE1 (GST-Ct) measured by a sensitive microtiter plate binding assay. Further amino-terminal truncation of CAII by 17 or 24 residues caused a loss of binding. The homologous isoform CAI does not bind AE1, despite having 60% sequence identity to CAII. One major difference between the two CA isoforms, within the amino-terminal region, is a high content of histidine residues in CAII (His3, -4, -10, -15, -17) not found in CAI. Mutation of pairs of these histidines (and one lysine) in CAII to the analogous residues in CAI (H3P/H4D or K9D/H10K or H15Q/H17S), or combinations of these various double mutants, did not greatly affect binding between GST-Ct and the mutant CAII. However, when all six of the targeted CAII residues were mutated to the corresponding sequence in CAI, binding of GST-Ct was lost. These results indicate that the AE1 binding site is located within the first 17 residues of CAII, and that the interaction is mediated by electrostatic interactions involving histidine and/or lysine residues. Further specificity for the interaction of AE1 and CAII is provided by a conserved leucine residue (L886) in AE1 that, when mutated to alanine, resulted in loss of GST-Ct binding to immobilized CAII. The binding of the basic amino-terminal region of CAII to an acidic Ct in AE1 provides a structural basis for linking bicarbonate transport across the cell membrane to intracellular bicarbonate metabolism.     

Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.     

Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause approximately 2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALS-related SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133 Delta) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.     

Metal Binding Is Critical for the Folding and Function of Laminin Binding Protein,Lmb of Streptococcus agalactiae

Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb) and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.     

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.     

Crystallographic and NMR investigation of cobalt-substituted amicyanin

Cobalt(II) amicyanin was prepared by replacing the copper of the type I copper protein amicyanin from Paracoccus denitrificans with cobalt. The structure of the protein and the metal center have been characterized by X-ray crystallography and paramagnetic NMR spectroscopy. The crystal structure indicates that Met98, which provides an axial sulfur ligand in native amicyanin, is no longer bound to the metal in cobalt(II) amicyanin and that a water molecule is recruited from solvent to form the fourth metal ligand. This results in a tetrahedral coordination geometry for the cobalt ion. NMR studies in solution also indicate that the side chain of the methionine residue interacts less strongly with the metal in P. denitrificans amicyanin than in Paracoccus versutus amicyanin. The cobalt(II) amicyanin crystal structure is different from that of cobalt-substituted azurin in which the carbonyl of a glycine residue provides this equivalent ligand. In cobalt(II) amicyanin that residue is a proline, for which the oxygen is structurally inaccessible, so that the water occupies the position held by the glycine carbonyl in cobalt(II) azurin. Such a metal coordination involving water has not previously been reported for a native or metal-substituted type I copper protein.     

Chemical modification of prothrombin fragment 1: documentation of sequential, two-stage loss of protein function

The amino groups of prothrombin fragment 1 (amino acids 1-156 of prothrombin) were derivatized by acetylation, amidination, and reductive methylation. Conditions that caused complete acetylation of protein amino groups produced a fragment 1 derivative which no longer displayed a metal ion dependent intrinsic fluorescence change and had lost its membrane binding capability as well. However, when derivatized in the presence of calcium ions, extensive acetylation yielded a product that underwent protein fluorescence quenching at metal ion concentrations similar to those observed for the native protein. This derivative bound to membranes in a calcium-dependent manner with only a small reduction in affinity. Several results showed the existence of a partially functional protein that was characterized by a high degree of calcium-dependent protein fluorescence quenching but which had a requirement for 10-fold higher calcium concentration. This derivative was produced by partial acetylation (greater than 3 equiv) of metal-free protein. This partially acetylated protein had greatly diminished membrane binding. The calcium-protected amino group, therefore, was among the most reactive acetylation sites in the metal-free protein. The second site, responsible for abolishing all metal ion induced fluorescence change, was resistant to acetylation and became derivatized at the last stages of amino group acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structures of the reduced and Cu(I) bound forms of the first metal binding sequence of ATP7A associated with Menkes disease

The coding sequence for the first N-terminal copper binding motif of the human Menkes disease protein (MNK1; residues 2-79) was synthesized, cloned, and expressed in bacteria for biochemical and structural studies. MNK1 adopts the betaalphabetabetaalphabeta fold common to all the metal binding sequences (MBS) found in other metal transport systems (e.g., the yeast copper chaperone for superoxide dismutase CCS, the yeast copper chaperone ATX1 bound to Hg(II), and most recently Cu(I), the bacterial copper binding protein, CopZ, and the bacterial Hg(II) binding protein MerP), although substantial differences were found in the metal binding loop. Similar to ATX1, MNK1 binds Cu(I) in a distorted linear bicoordinate geometry. As with MerP, MNK1 has a high affinity for both Hg(II) and Cu(I), although it displays a marked preference for Cu(I). In addition, we found that F71 is a key residue in the compact folding of MNK1, and its mutation to alanine results in an unfolded structure. The homologous residue in MerP has also been mutated with similar results. Finally, to understand the relationship between protein folding and metal affinity and specificity, we expressed a chimeric MBS with the MNK1 protein carrying the binding motif of MerP (CAAC-MNK1); this chimeric protein showed differences in structure and the dynamics of the binding site that may account for metal specificity.     

Probing metal ion substrate-binding to the E. coli ZitB exporter in native membranes by solid state NMR

Metal ion homeostasis is important for healthy cell function and is regulated by metal ion transporters and chaperones. To explore metal ion binding to membrane transport proteins we have used cadmium-113 as a solid state NMR probe of the Escherichia coli zinc exporter ZitB present in native membrane preparations. Competition experiments with other metal ions indicated that nickel and copper are also able to bind to this protein. Metal ion uptake studies were also performed using ZitB-reconstituted into proteoliposomes for a well established fluorescence assay. The results of both the solid state NMR and the uptake studies demonstrate that ZitB is potentially capable of transporting not only zinc but also cadmium, nickel and copper. The solid state NMR approach therefore offers great potential for defining the substrate spectrum of metal ion transporter proteins in their native membrane environments. Further, it should be useful for functional dissection of transporter mechanisms by facilitating the identification of functional residues by mutational studies.