Duncan E. K. Sutherland

Noncooperative cadmium(II) binding to human metallothionein 1a

The two-domain (beta alpha) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain beta alpha-rhMT and the isolated beta-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd(3)-beta-rhMT and Cd(7)-beta alpha-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd(4)-beta- and Cd(8)-beta alpha-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins.

Noncooperative Metalation of Metallothionein 1a and Its Isolated Domains with Zinc

Mammalian metallothioneins (MTs) are a family of small cysteine-rich proteins capable of binding 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal β-domain that binds 3 Zn(2+) or Cd(2+) and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). MT has been implicated in a number of physiological functions, including metal ion homeostasis, toxic metal detoxification, and as a protective agent against oxidative stress. Conventionally, MT has been understood to coordinate metal ions in a cooperative fashion. Under this mechanism of metalation, the only species of biological relevance would be the metal-free (apo-) form of the protein and the fully metalated (holo-) form of the protein. However, an increasing body of evidence suggests that metalation occurs in a noncooperative manner. If this latter mechanism is correct, then partially metalated forms of the protein will be stable and able to take part in cellular chemistry. We report in this paper conclusive evidence that shows that biologically essential zinc binds to MT in a noncooperative manner. In addition, we report for the first time the stability of a Zn(5)-MT species. The implications of these findings are discussed in terms of the mechanism of metalation.

The Ni(II)-binding properties of the metallochaperone SlyD.

Metallochaperones are essential for the safe and targeted delivery of necessary yet toxic metal cofactors to their respective protein partners. In this study we examine the nickel-binding properties of the Escherichia coli protein SlyD, a factor that contributes to optimal nickel accumulation in this organism. This protein is also required for E. coli energy metabolism because it participates in the nickel insertion step during [Ni-Fe]-hydrogenase metallocenter assembly. Our study demonstrates that SlyD is a multiple nickel ion binding protein. The analysis of noncovalent metal-protein complexes via electrospray ionization mass spectrometry revealed that SlyD binds up to seven nickel ions in a noncooperative manner with submicromolar affinity (<2 microM, upper limit) and that the protein exists in a dynamic mixture of metalloforms that is dependent on the availability of nickel ions in solution. Structural analysis indicates that this metallochaperone undergoes small but distinct changes in the structure upon metal binding and that the nickel-binding sites are assembled through beta-turn formation. Although the C-terminal metal-binding domain is primarily responsible for metal chelation, we find that metal binding also perturbs the structure of the N-terminal domains. An investigation of the nickel sites by using X-ray absorption spectroscopy shows that SlyD binds nickel ions by adapting several different geometries and coordination numbers. Finally, the characterization of SlyD mutants demonstrates that the cysteine residues in the C-terminal domain confer tighter affinity as well as increased binding capacity to SlyD. On the basis of the presented data a model for nickel binding to SlyD as well as its role in nickel homeostasis is discussed.

Single-Domain Metallothioneins: Evidence of the Onset of Clustered Metal Binding Domains in Zn-rhMT 1a

Mammalian metallothioneins bind up to seven Zn(2+) ions in two distinct domains: an N-terminal β-domain that binds three Zn(2+) ions and a C-terminal α-domain that binds four Zn(2+) ions. Domain specificity has been invoked in the metalation mechanism with cluster formation and bridging of the 20 Cys residues taking place prior to saturation with seven Zn(2+) ions. We report a novel experiment that examines Zn(2+) metalation by exploiting the expected decrease in K(F) at the onset of clustering using electrospray ionization mass spectrometry (ESI-MS). During the titration with Zn(2+), the ESI-MS data show that several metalated species coexist until the fully saturated proteins are formed. The relative Zn binding affinities of the seven total sites in the α- and β-fragments were determined through direct competition for added Zn(2+). The K(F) values for each Zn(2+) are expected to decrease as a function of the remaining available sites and the onset of clustering. Analysis shows that Zn(2+) binds to β-rhMT with a greater affinity than α-rhMT. The incremental distribution of Zn(2+) between the competing fragments and apo-βα-rhMT (essentially three and four sites competing with seven sites) identifies the exact point at which clustering begins in the full protein. Analysis of the speciation data shows that Zn(5)-MT forms before clustering begins. This means that all 20 Cys residues of apo-βα-rhMT are bound terminally to Zn(2+) as [Zn(Cys)(4)](2-) units before clustering begins; there is no domain preference in this first metalation stage. Preferential binding of Zn(2+) to β- and α-rhMT at the point where βα-rhMT must form clusters is caused by a significant decrease in the affinity of βα-rhMT for further Zn(2+). The single-domain Zn(5)-rhMT, in which there are no exposed cysteine sulfurs, is a key component of the metalation pathway because the lower affinities of the two clustered Zn(2+) ions allow donation to apoenzymes.

Supermetalation of the beta domain of human metallothionein 1a.

Metallothionein has been implicated in a number of functions, including toxic metal detoxification, as a metal chaperone and in metal ion homeostasis. In this paper, we demonstrate that the beta domain of human metallothionein 1a, well-known to bind three Zn(2+) or Cd(2+) ions with nine cysteinyl sulfurs, is also capable of binding an additional Cd(2+) ion, leading to the formation of the supermetalated Cd(4)-beta-rhMT 1a. This intermediate, either by itself or in concert with the alpha domain of human metallothionein, is a likely model for metal exchange with the apoenzyme, which is one of the key roles of metallothionein. Through electrospray ionization (ESI) mass spectrometry and circular dichroism (CD) and ultraviolet (UV) spectroscopy, we show that the addition of 4.4 molar equiv of CdSO(4) to a solution of Cd(3)-beta-rhMT 1a leads to the complete conversion to Cd(4)-beta-rhMT 1a. ESI mass spectrometry was used to determine the exact speciation of beta-rhMT 1a. While the UV absorption spectrum increased slightly, the CD spectrum of Cd(4)-beta-rhMT 1a showed significant changes with the appearance of a sharp monophasic peak at 252 nm in contrast to the derivative-shaped envelope of the Cd(3)-beta-rhMT 1a species [peak extrema at (+)262 and (-)236 nm], indicating disruption of the exciton coupling in the metal-thiolate cluster. Additionally, both direct and indirect (113)Cd nuclear magnetic resonance (NMR) spectra of the Cd(3)-beta-rhMT 1a and Cd(4)-beta-rhMT 1a species were recorded. The (113)Cd NMR spectrum of Cd(4)-beta-rhMT 1a contained four cadmium peaks in the tetrahedral thiolate region at 688.8, 650.3, 635.9, and 602.5 ppm. This represents the first report of both NMR data for isolated Cd(3)-beta-rhMT 1a and supermetalated Cd(4)-beta-rhMT 1a.

Cu(I) binding properties of a designed metalloprotein

The Cu(I) binding properties of the designed peptide C16C19-GGY are reported. This peptide was designed to form an alpha-helical coiled-coil but modified to incorporate a Cys-X-X-Cys metal-binding motif along its hydrophobic face. Absorption, emission, electrospray ionization mass spectrometry (ESI-MS), and circular dichroism (CD) experiments show that a 1:1 Cu-peptide complex is formed when Cu(I) is initially added to a solution of the monomeric peptide. This is consistent with our earlier study in which the emissive 1:1 complex was shown to exist as a peptide tetramer containing a tetranuclear copper cluster Kharenko et al. (2005) [11]. The presence of the tetranuclear copper center is now confirmed by ESI-MS which along with UV data show that this cluster is formed in a cooperative manner. However, spectroscopic titrations show that continued addition of Cu(I) results in the occupation of a second, lower affinity metal-binding site in the metallopeptide. This occupancy does not significantly affect the conformation of the metallopeptide but does result in a quenching of the 600nm emission. It was further found that the exogenous reductant tris(2-carboxyethyl)phosphine (TCEP) can competitively inhibit the binding of Cu(I) to the low affinity site of the peptide, but does not interact with Cu(I) clusters.

Modeling the Zn2+ and Cd2+ metalation mechanism in mammalian metallothionein 1a

Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal β-domain that binds 3 Zn(2+) or Cd(2+), and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). Although stepwise metalation to the saturated M(7)-MT (where M=Zn(2+) or Cd(2+)) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (M(n)-MT, n=0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K(1)K(2)>K(3)>···>K(7)). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn(2+) and Cd(2+) are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn(2+) and Cd(2+) binding to rhMT 1a.

Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed.

Stabilization of protein structure through π-π interaction in the second coordination sphere of pseudoazurin.

Noncovalent, weak interactions in the second coordination sphere of the copper active site of Pseudoazurin (PAz) from Achromobacter cycloclastes were examined using a series of Met16X variants. In this study, the differences in protein stability due to the changes in the nature of the 16th amino acid (Met, Phe, Val, Ile) were investigated by electrospray ionization mass spectrometry (ESI‐MS) and far‐UV circular dichroism (CD) as a result of acid denaturation. The percentage of native states (folded holo forms) of Met16Phe variants was estimated to be 75% at pH 2.9 although the wild‐type (WT), Met16Val and Met16Ile PAz, became completely unfolded. The high stability under acidic conditions is correlated with the result of the active site being stabilized by the aromatic substitution of the Met16 residue. The π–π interaction in the second coordination sphere makes a significant contribution to the stability of active site and the protein matrix.

Metal selectivity of the E. coli nickel metallochaperone, SlyD

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed.