Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies.

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.

Bi(III) Binding Stoichiometry and Domain-Specificity Differences Between Apo and Zn(II)-bound Human Metallothionein 1a.

Bismuth is a xenobiotic metal with a high affinity to sulfur that is used in a variety of therapeutic applications. Bi(III) induces the cysteine-rich metallothionein (MT), a protein known to form two-domain cluster structures with certain metals such as Zn(II), Cd(II), or Cu(I). The binding of Bi(III) to MTs has been previously studied, but there are conflicting reports on the stoichiometry and binding pathway, which appear to be highly dependent on pH and initial metal-loading status of the MT. Additionally, domain specificity has not been thoroughly investigated. In this paper, ESI-MS was used to determine the binding constants of [Bi(EDTA)]- binding to apo-MT1a and its individual αMT fragment. The results were compared to previous experiments using ßMT1a and ßαMT3. Domain specificity was investigated using proteolysis methods and the initial cooperatively formed Bi2MT was found to bind to cysteines that spanned across the traditional metal binding domain regions. Titrations of [Bi(EDTA)]- into Zn7MT were performed and were found to result in a maximum stoichiometry of Bi7MT, contrasting the Bi6MT formed when [Bi(EDTA)]- was added to apo-MT. These results show that the initial structure of the apo-MT determines the stoichiometry of new incoming metals and explains the previously observed differences in stoichiometry.

Structural motifs in the early metallation steps of Zn(II) and Cd(II) binding to apo-metallothionein 1a.

Many proteins require a metal cofactor to function and these metals are often involved in the protein folding process. The protein metallothionein (MT) has a dynamic structure capable of binding to a variety of metals with different stoichiometries. The most well-understood structure is the seven-metal, two domain structure formed upon metallation using Zn(II) or Cd(II). However, the partially metallated states and the pathways to form these clusters are less well-understood, although it is known that the pathways are pH dependent. Using stopped flow methods, it is shown that the metallation rates of the less cooperative Zn(II) binding pathway is much more impacted by low pH conditions that that of the more cooperative Cd(II) binding pathway. Electrospray ionization mass spectrometry (ESI-MS) methods reveal specific mixtures of bridging and terminally bound MxSy structures form in the first few metallation steps. Using a combination of methods, the data show that the result of unfolding this intrinsically disordered apo-MT structure using guanidinium chloride is that the formation of preliminary bridging structures that form in the first few metallation steps is impeded. The data show that more terminally bound structures form. Our conclusion is that the compact conformation of the native apo-MT at physiological pH allows for rapid formation of complex metal-thiolate structures with high affinity that provides protection from oxidation, a function that is suppressed upon unfolding. Overall, these results highlight both the importance of the apo-MT structure in the metallation pathway, but also the differences in Zn(II) and Cd(II) binding under different conditions.

Structural Role of Cadmium and Zinc in Metallothionein Oxidation by Hydrogen Peroxide: The Resilience of Metal-Thiolate Clusters.

Oxidative stress is a state involving an imbalance of reactive oxygen species in a cell and is linked to a variety of diseases. The metal-binding protein metallothionein (MT) may play a role in protection due to its high cysteine content. Many studies have shown that oxidative stress will cause MT to both form disulfide bonds and release bound metals. However, studies on the more biologically relevant partially metalated MTs have been largely neglected. Additionally, most studies to date have used spectroscopic methods that cannot detect specific intermediate species. In this paper, we describe the oxidation and the subsequent metal displacement pathway of fully and partially metalated MTs with hydrogen peroxide. The rates of the reactions were monitored using electrospray ionization mass spectrometry (ESI-MS) techniques, which resolved and characterized the individual intermediate Mx(SH)yMT species. The rate constants were calculated for each species formation. Through ESI-MS and circular dichroism spectroscopy, it was found that the three metals in the ß-domain were the first to be released from the fully metalated MTs. The Cd(II) in the partially metalated Cd(II)-bound MTs rearranged to form a protective Cd4MT cluster structure upon exposure to oxidation. The partially metalated Zn(II)-bound MTs oxidized at a faster rate as the Zn(II) did not rearrange in response to oxidation. Additionally, density functional theory calculations showed that the terminally bound cysteines were more negative and thus more susceptible to oxidation than the bridging cysteines. The results of this study highlight the importance of metal-thiolate structures and metal identity in MT's response to oxidation.

Apo-metallothionein-3 cooperatively forms tightly compact structures under physiological conditions.

Metallothioneins (MTs) are essential mammalian metal chaperones. MT isoform 1 (MT1) is expressed in the kidneys and isoform 3 (MT3) is expressed in nervous tissue. For MTs, the solution-based NMR structure was determined for metal-bound MT1 and MT2, and only one X-ray diffraction structure on a crystallized mixed metal-bound MT2 has been reported. The structure of solution-based metalated MT3 is partially known using NMR methods; however, little is known about the fluxional de novo apo-MT3 because the structure cannot be determined by traditional methods. Here, we used cysteine modification coupled with electrospray ionization mass spectrometry, denaturing reactions with guanidinium chloride, stopped-flow methods measuring cysteine modification and metalation, and ion mobility mass spectrometry to reveal that apo-MT3 adopts a compact structure under physiological conditions and an extended structure under denaturing conditions, with no intermediates. Compared with apo-MT1, we found that this compact apo-MT3 binds to a cysteine modifier more cooperatively at equilibrium and 0.5 times the rate, providing quantitative evidence that many of the 20 cysteines of apo-MT3 are less accessible than those of apo-MT1. In addition, this compact apo-MT3 can be identified as a distinct population using ion mobility mass spectrometry. Furthermore, proposed structural models can be calculated using molecular dynamics methods. Collectively, these findings provide support for MT3 acting as a noninducible regulator of the nervous system compared with MT1 as an inducible scavenger of trace metals and toxic metals in the kidneys.

Structurally restricted Bi(III) metallation of apo-ßMT1a: metal-induced tangling.

Non-toxic bismuth salts are used in anti-ulcer medications and to protect against nephrotoxicity from anticancer drugs. Bismuth salts also induce metallothionein (MT), a metal-binding protein that lacks a formal secondary structure. We report the impact on the metallation properties of Bi(III) to the 9-cysteine ß fragment of MT as a function of cysteine accessibility using electrospray ionization mass spectrometry. At pH 7.4, Bi2ßMT formed cooperatively. Cysteine modification shows that each Bi(III) was terminally bound to three cysteinyl thiolates. Non-cooperative Bi(III) binding was observed at pH 2.3, where cysteine accessibility is increased. However, competition from H4EDTA inhibited Bi(III) binding. When GdmCl, a well-known denaturing agent, was used to increase cysteine accessibility of the apoßMT at pH 7.4, a greater fraction of Bi3ßMT formed using all nine cysteines. The change in binding profile and equilibrium of Bi2ßMT was determined as a function of acidification, which changed as a result of competition with H4EDTA. There was no Bi(III) transfer between Bi2ßMT, Cd3ßMT, and Zn3ßMT. This lack of metal exchange and the resistance towards binding the third Bi(III) suggest a rigidity in the Bi2ßMT binding sites that inhibits Bi(III) mobility. These experiments emphasize the conformational control of metallation that results in substantially different metallated products: at pH 7.4 (many cysteines buried) Bi2ßMT, whereas at pH 7.4 (all cysteines accessible) enhanced formation of Bi3ßMT. These data suggest that the addition of the first two Bi(III) crosslinks the protein, blocking access to the remaining three cysteines for the third Bi(III), as a result of tangle formation.

The pathways and domain specificity of Cu(I) binding to human metallothionein 1A.

Copper is an essential element, but as a result of numerous adverse reactions, it is also a cellular toxin. Nature protects itself from these toxic reactions by binding cuprous copper to chaperones and other metalloproteins. Metallothionein has been proposed as a storage location for Cu(i) and potentially as the donor of Cu(i) to copper-dependent enzymes. We report that the addition of Cu(i) to apo recombinant human metallothionein 1a cooperatively forms a sequential series of Cu(i)-cysteinyl thiolate complexes that have specific Cu(i) : MT stoichiometries of 6 : 1, 10 : 1, and finally 13 : 1. The individual domain Cu : SCys stoichiometries were determined as Cu6S9 (for 6 : 1), Cu6S9 + Cu4S6 (for 10 : 1), and Cu6S9 + Cu7S9 (for 13 : 1) based on the number of modified free cysteines not involved in Cu(i) binding. The stoichiometries are associated with Cu-SCys cluster formation involving bridging thiols in the manner similar to the clusters formed with Cd(ii) and Zn(ii). The locations of these clustered species within the 20 cysteine full protein were determined from the unique speciation profiles of Cu(i) binding to the ß and α domain fragments of recombinant human metallothionein 1a with 9 and 11 cysteines, respectively. Competition reactions using these domain fragments challenged Cu(i) metallation of the ßα protein, allowing the sequence of cluster formation in the full protein to be determined. Relative binding constants for each Cu(i) bound are reported. The emission spectra of the Cu4S6, Cu6S9, and Cu7S9 clusters have unique λmax and phosphorescent lifetime properties. These phosphorescent data provide unambiguous supporting evidence for the presence of solvent shielded clusters reported concurrently by ESI-MS. Simulated emission spectra based on the cluster specific emission profiles matched the experimental spectra and are used to confirm that the relative concentrations seen by ESI-MS are representative of the solution. Our results suggest that the availability of a series of sequential Cu(i)-thiolate clusters provides flexibility as a means of protecting the cell from toxicity while still allowing for homeostatic control of the total copper content in the cell. This mechanism provides a dynamic and reactive method of reducing the cellular free copper concentrations.

Interplay between Carbonic Anhydrases and Metallothioneins: Structural Control of Metalation.

Carbonic anhydrases (CAs) and metallothioneins (MTs) are both families of zinc metalloproteins central to life, however, they coordinate and interact with their Zn2+ ion cofactors in completely different ways. CAs and MTs are highly sensitive to the cellular environment and play key roles in maintaining cellular homeostasis. In addition, CAs and MTs have multiple isoforms with differentiated regulation. This review discusses current literature regarding these two families of metalloproteins in carcinogenesis, with a dialogue on the association of these two ubiquitous proteins in vitro in the context of metalation. Metalation of CA by Zn-MT and Cd-MT is described. Evidence for protein-protein interactions is introduced from changes in metalation profiles of MT from electrospray ionization mass spectrometry and the metalation rate from stopped-flow kinetics. The implications on cellular control of pH and metal donation is also discussed in the context of diseased states.

Metallothionein Cd4S11 cluster formation dominates in the protection of carbonic anhydrase.

Metallothioneins (MTs) are ubiquitous proteins vital for essential metal homeostasis and heavy metal detoxification. The twenty-cysteinyl mammalian metallothioneins protect the proteome by sequestering heavy metals into thermodynamically stable metal thiolate structures when metalated with seven Cd2+. At physiological pH, the first metal (M) thiolate (SCys) structures formed involve M(SCys)4 terminal thiolates. With higher metal loading, M4(SCys)11 and M3(SCys)9 clusters form. As a regulator of the metallome, it is necessary to understand metal sequestration properties of MT in solution with other metalloproteins. We report that the association between apo-MT and apo-carbonic anhydrase (CA) enhances the formation of the protective mode of MT, in which Cd4(SCys)11-clusters form at much lower concentration levels than for the free apo-MT at physiological pH. Using stopped-flow kinetics and electrospray ionization mass spectrometry, we quantified this protective effect, determining that it is significant at pH 7.4, but the effect diminishes at pH 5.0. We report for the first time, the absolute stepwise binding constants of Cd2+ binding to human MT1a both in the presence and absence of CA through calibration by the known binding constant of Cd2+ to bovine CA. We report that this protein association affects the Cd2+ metalation rates of MT. The data support the physiological role of MTs as protectors of the metalloproteome from the toxic effects of Cd2+.

pH dependence of the non-cooperative binding of Bi3+ to human apo-metallothionein 1A: kinetics, speciation, and stoichiometry.

Bismuth is a well-known therapeutic agent that is used primarily for treatment against peptic ulcers. It has also had success in protecting against nephrotoxicity caused by the anticancer compound cisplatin by inducing the liver and kidney metalloprotein, metallothionein (MT) that then binds to the cisplatin. MT is a small, ubiquitous protein that binds monovalent, divalent, and trivalent metals using its abundant cysteine thiols (20 cysteines in the mammalian protein). It is important in the understanding of both these therapeutic applications to explore in detail the earliest stages of MT binding to bismuth salts. In this paper, we explored the binding of [Bi(cit)]- and [Bi(EDTA)]- to apo-MT 1a as the most basic of binding motifs. It was found that both Bi3+ salts bound in a non-cooperative stepwise manner to terminal cysteinal thiolates at pH 2.6, 5.0, and 7.4. We report that [Bi(EDTA)]- only binds stepwise up to Bi6MT, whereas [Bi(cit)]- forms up to Bi8MT, where the 7th and 8th Bi3+ appear to be adducts. Stepwise speciation analysis provided the 7 binding constants that decreased systematically from K1 to K7 indicating a non-cooperative binding profile. They are reported as log K1 = 27.89, log K2 = 27.78, log K3 = 27.77, log K4 = 27.62, log K5 = 27.32, log K6 = 26.75, and log K7 = 26.12, with log K[Bi(cit)]- determined to be 24.17. Cysteine modifications with benzoquinone and iodoacetamide revealed that when apoMT is fully metallated with Bi3+ there are two free cysteines, meaning 18 cysteines are used in binding the 6 Bi3+. Kinetic studies showed that [Bi(EDTA)]- binds very slowly at pH 2.6 (k = 0.0290 × 106 M-1 s-1) and approximately 2000 times faster at pH 7.4 (k = 66.5 × 106 M-1 s-1). [Bi(cit)]- binding at pH 2.6 was faster than [Bi(EDTA)]- (k = 672 × 106 M-1 s-1) at either pH level. The data strongly support a non-clustered binding motif, emphasizing the non-traditional pathway reported previously for As3+.

Kinetics of competitive Cd2+ binding pathways: the realistic structure of intrinsically disordered, partially metallated metallothioneins.

The 20-cysteine mammalian metallothioneins are considered to be central to the homeostatic control of the essential metals Zn(ii) and Cu(i) and, as part of their metal-loaded status, play a role in reversing oxidative stress. Native apo-MT does not adopt a well-known structural motif, being described as a random-coil or intrinsically-disordered. Conclusions reached from a combination of ESI-mass spectral charge states, As(iii) metallation of apo-MT at low pH, from molecular dynamic calculations and from metallation of the α-domain fragment, suggest that in fact the native apo-MT adopts a structure that is highly efficient towards metallation at physiological pH. The results in this paper show that the initial (M < 5) Cd(ii) metallation at physiological pH takes place to form structures based on isolated Cd(SCYS)4 units, beads. At pH 5, cysteine bridged Cd4(SCYS)11 clusters form. ESI-mass spectral profile of cysteine modification of apo-MT at physiological pH shows that it is folded, whereas in the presence of 3 M guandinium hydrochloride the apo-MT is unfolded. Stopped flow kinetic studies of the Cd(ii) metallation shows that the reaction is much slower for the unfolded vs. the folded apo-MT for formation of either beads or clusters. Metallation is also much slower for the formation of clusters than the formation of beads. These results are first to quantify the presence of structure in native apo-MT in terms of the critical metallation properties. The implications of this study suggest that oxidation of apo-MT due to ageing or other agent will negatively impact the metallation process for essential metals.