Unexpected Interactions of the Cyanobacterial Metallothionein SmtA with Uranium

Abstract

Molecules for remediating or recovering uranium from contaminated environmental resources are of high current interest, with protein-based ligands coming into focus recently. Metallothioneins either bind or redox-silence a range of heavy metals, conferring protection against metal stress in many organisms. Here, we report that the cyanobacterial metallothionein SmtA competes with carbonate for uranyl binding, leading to formation of heterometallic (UO2)(n)Zn4SmtA species, without thiol oxidation, zinc loss, or compromising secondary or tertiary structure of SmtA. In turn, only metalated and folded SmtA species were found to be capable of uranyl binding. (1)H NMR studies and molecular modeling identified Glu34/Asp38 and Glu12/C-terminus as likely adventitious, but surprisingly strong, bidentate binding sites. While it is unlikely that these interactions correspond to an evolved biological function of this metallothionein, their occurrence may offer new possibilities for designing novel multipurpose bacterial metallothioneins with dual ability to sequester both soft metal ions including Cu(+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+) and hard, high-oxidation state heavy metals such as U(VI). The concomitant protection from the chemical toxicity of uranium may be valuable for the development of bacterial strains for bio-remediation.

Uranyl Binding to Proteins and Structural-Functional Impacts

“…Similarly, UO2 2+ may bind to these metalloproteins, depending on the properties of their surfaces, and cause structural and functional consequences. Acharya and Blindauer reported that UO2 2+ can bind to the cyanobacterial metallothionein SmtA, and form a heterometallic (UO2 2+ )nZn4SmtA species, without alteration of the native Zn4Cys9His2 cluster [49]. 1 H-NMR and molecular modeling studies In addition to a single metal ion or metal cofactor, metalloproteins may harbor multiple metal ions forming metal clusters that play either structural roles, electron-transfer, or catalysis functions [47].…”

“…Thirdly, although some structural features of uranyl-protein complexes are well-documented [7][8][9], it is still needed to obtain more structural information for uranyl-protein complexes, not only in solid state (such as X-ray crystal structure [10]), but also in solution state (such as NMR structure [49]), for a deep understanding of uranyl-induced conformational changes.…”

Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins

“…The lack of a rigid structure allows MTs to coordinate a number of metals including essential metals like zinc and copper [11], toxic metals like cadmium [12] and mercury [13], and exotic metals like uranium [14] and technetium [15]. This flexibility, while fundamental to their function, makes MTs extremely difficult to characterize structurally.…”

Formation of oxidative and non-oxidative dimers in metallothioneins: Implications for charge-state analysis for structural determination

“…Cadmium supplementation has also been shown to produce MT dimers isolated from rabbit liver . Significant amounts of dimerization has been reported for the interaction of bacterial MT with uranyl salts (UO 2 2+ ) where the stable dimer product was subsequently characterized by proteolytic digestion and analysis by Ellman's method using 5,5′‐dithiobis(2‐nitrobenzoic acid) to quantify free thiols . The bacterial MT was completely oxidized and formed significant numbers of ions in the ESI‐MS spectrum, some of which overlapped with the monomer .…”

“…Significant amounts of dimerization has been reported for the interaction of bacterial MT with uranyl salts (UO 2 2+ ) where the stable dimer product was subsequently characterized by proteolytic digestion and analysis by Ellman's method using 5,5′‐dithiobis(2‐nitrobenzoic acid) to quantify free thiols . The bacterial MT was completely oxidized and formed significant numbers of ions in the ESI‐MS spectrum, some of which overlapped with the monomer . This overlap is crucial, as it may confound one of the few techniques available for structural analysis of the dynamic and difficult to characterize MT.…”