Non-coordinative metal selectivity bias in human metallothioneins metal–thiolate clusters

Abstract

Mammalian metallothioneins (MT-1 through MT-4) are a class of metal binding proteins containing two metal-thiolate clusters formed through the preferential coordination of d10 metals, Cu(I) and Zn(II), by 20 conserved cysteine residues located in two protein domains. MT metalation (homometallic or heterometallic Zn(II)/Cu(I) species) appears to be isoform specific and controlling zinc and copper concentrations to perform specific and distinct biological functions. Structural and functional relationships, and in vivo metalation studies, identified evolutionary features defining the metal-selectivity nature for MTs. Metallothionein-3 (MT-3) has been shown to possess the most pronounced Cu-thionein character forming Cu(I)-containing species more favorably than metallothionein-2 (MT-2), which possesses the strongest Zn-thionein character. In this work, we identify isoform-specific determinants which control metal binding selectivity bias in different MTs isoforms. By studying the reactivity of Zn7MT-2, Zn7MT-3 and Zn7MT-3 mutants towards Cu(II) to form Cu(I)4Zn4MTs, we have identified isoform-specific key non-coordinating residues governing folding/outer sphere control of metal selectivity bias in MTs metal clusters. By mutating selected residues and motifs in MT-3 to the corresponding MT-2 amino acids, we dissected key roles in modulating cluster dynamic and metal exchange rates, in increasing the Cu(I)-affinity in MT-3 N-terminal β-domain and/or modulating the higher stability of the Zn(II)-thiolate cluster in MT-2 β-domain. We thus engineered MT-3 variants in which the copper-thionein character is converted into a zinc-thionein. These results provide new insights into the molecular determinants governing metal selectivity in metal-thiolate clusters.

Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells

“…The low fractional saturation of crisp-17 indicates that ATP7A-null cells retain the ability to buffer copper within the low attomolar range, despite the large increase in total cellular copper. This observation is consistent with recent estimates of the Cu(I) binding affinity of metallothionein (29,32), which is up-regulated in Menkes mutant cells and has been implicated as the major copper carrier (33). Although we found strong induction of metallothionein transcription in ATP7A-null cells as expected (SI Appendix, Fig.…”

“…While we do not exclude that direct buffering of Cu(I) by GSH may come to play under acutely toxic conditions, such as ionophore-mediated rapid influx of Cu(II) or partial oxidation of cellular thiols by DTDP, the ability of GSH-Cu(I) complexes to catalyze superoxide formation in vitro (37) implies that cellular Cu(I) levels should be maintained below the threshold for GSH cluster assembly to avoid toxicity in an oxygenated atmosphere. In contrast, the tetranuclear Cu(I)-cluster formed in the β-domain of metallothioneins is redox-inert in air (29), thus rendering metallothionein a safe Cu(I)-storage site, consistent with its up-regulation in ATP7A-null fibroblasts. Nevertheless, copper-supplemented 3T3 cells maintain tight control over Cu(I) availability without up-regulating metallothionein, thus suggesting the involvement of a more complex polydisperse buffer, potentially including yet-to-be-discovered thiol ligands.…”

The Glutathione/Metallothionein System Challenges the Design of Efficient O2‐Activating Copper Complexes

“…Forschungsartikel mainly Cu I 4 -MT was formed after 4hours incubation, depending on the copper complex, contributions of other Cu I 4 -MT dependent complexes (such as ternary complexes of Cu I 4 -MT with GSH and/or ligands) were observed upon mixing. [16,18] Concerning the stability of the studied copper complexes against copper transfer to cytosolic-relevant concentrations of GSH and Zn 7 -MT,t he following observations can be made: i) Cu I -(BCS) 2 complex dissociates within the mixing-time and Cu I binds to MT.B CS and derivatives are some of the strongest Cu I chelators used in biology.H owever,a t1 0mm concentration, the complex is not thermodynamically stable enough to resist to Cu I transfer to MT.Ahigher concentration of hundreds of mm BCS would be needed to compete for Cu I with MT.H owever, such high concentrations are difficult to reach and less relevant from ad rug point of view.i i) Cu II -(Phen) 2 and Cu II -(5,5'-DmBipy) 2 also dissociate within mixing-time.T his is in line with their very fast reduction to Cu I -(Phen) 2 and Cu I -(5,5'-DmBipy) 2 and with the lower thermodynamic stability of the Cu I -complexes compared to Cu I -MT complex (logb 2 [Cu I -(Phen) 2 ] = 15.8). [27] Thus,C u II -(Phen) 2 and Cu II -(5,5'-DmBipy) 2 are rapidly reduced and Cu I is immediately transferred to MT.iii)Dissociation by reduction of Cu II -Dp44mT,C u II -gtsm, and Cu II -(APDTC) 2 is slower compared to Cu II -(Phen) 2 and Cu II -(5,5'-DmBipy) 2, with t 1/2 of approximately 4, 50, and 20 min, respectively (Table 1).…”

“…MTs are very strong Cu I chelators (logK of [19][20][21]. [16] Based on these values,acopper complex must have ahigher Cu I -affinity than Cu I 4 -MT complex to be able to resist copper abstraction by MT.T on ote,M Ts can form ar edox-inert Cu I 4 S 5-7 cluster in their N-terminal b-domain by either direct Cu I binding or Cu II to Cu I reduction and concomitant formation of two disulfide bonds. [15] Under the condition used, with an excess of GSH, we expect the formed Cu I 4cluster to be present in afully reduced MT.Thus,inacellular context even bathocuproine-based Cu I complexes,s uch as Cu I -(BCS) 2 ,a re not stable at ar elatively high concentration of 10 mm.All the other tested copper complexes have alower Cu I -affinity.Only Cu II -(5,5'-DmBipy) 2 and Cu II -(Phen) 2 have roughly similar thermodynamic stability constants for both Cu I and Cu II redox states (logb 2 % 13-16).…”

“…[15] MTs are strong reducing agents,and hence able to reduce Cu II to Cu I and then bind it in the Cu I -thiolate cluster with concomitant formation of intramolecular disulfide bonds. [15,16] Another important compound is GSH, with concentrations ranging from 1-10 mm. [17] GSH can bind Cu I quite strongly (logK % 17) forming multinuclear clusters Cu I x -GSH y ,b ut under normal physiological conditions,asteady-state complex with Cu I is unlikely.…”