Interaction of Metallothionein-2 with Platinum-Modified 5′-Guanosine Monophosphate and DNA

Human metallothioneins (MTs), a family of cysteine- and metal-rich metalloproteins, play an important role in the acquired resistance to platinum drugs. MTs occur in the cytosol and the nucleus of the cells and sequester platinum drugs through interaction with their zinc−thiolate clusters. Herein, we investigate the ability of human Zn₇MT-2 to form DNA−Pt−MT cross-links using the cisplatin- and transplatin-modified plasmid DNA pSP73. Immunochemical analysis of MT-2 showed that the monofunctional platinum−DNA adducts formed DNA−cis/trans-Pt−MT cross-links and that platinated MT-2 was released from the DNA−trans-Pt−MT cross-links with time. The DNA−cis/trans-Pt−MT cross-links were also formed in the presence of 2 mM glutathione, a strong S-donor ligand. Independently, we used 5′-guanosine monophosphate (5′-GMP) platinated at the N7 position as a model of monofunctional platinum−DNA adducts. Comparison of reaction kinetics revealed that the formation of ternary complexes between Zn₇MT-2 and cis-Pt−GMP was faster than that of the trans isomer. The analysis of the reaction products with time showed that while the formation of ternary GMP−trans-Pt−MT complex(es) is accompanied by 5′-GMP release, a stable ternary GMP−cis-Pt−MT complex is formed. In the latter complex, a fast initial formation of two Pt−S bonds was followed by a slow formation of an additional Pt−S bond yielding an unusual Pt(II)S₃N coordination with N7-GMP as the only N-donor ligand. The ejection of negligible zinc from the zinc−thiolate clusters implies the initial formation of Zn−(μ-SCys)−Pt bridges involving the terminal thiolate ligands. The biological implications of these studies are discussed.