Crystallographic analysis of metal-ion binding to human Ubiquitin

The metal-binding ability of human ubiquitin (hUb) has been probed towards a selection of biologically relevant metal-ions and complexes. Different techniques have been used to obtain crystals suitable for crystallographic analysis. In a first type of experiments, crystals of hUb have been soaked in solutions containing copper(II) acetate and two metallodrugs: Zeise's salt and cisplatin. Zeise's salt is used in a test for hepatitis while cisplatin is one of the most powerful anticancer drugs in clinical use. Zeise's salt reacts smoothly with hUb crystals affording an adduct with three platinum residues per protein molecule: Pt3-hUb. In contrast, copper(II) acetate and cisplatin were found to be unreactive for contact times up to one hour and to cause degradation of the hUb crystals for longer times. In a second type of experiments, human ubiquitin was co-crystallized with solutions of copper(II) and zinc(II) acetate and with cisplatin. Zinc(II) acetate gives, at low metal-to-protein molar ratio, crystals containing one metal ion per three molecules of protein, Zn-hUb3 (already reported in a previous work), while at high metal-to-protein ratio (70:1) gives crystals containing three Zn(II) ions per protein molecule: Zn3-hUb. In contrast, once again, copper(II) acetate and cisplatin, even at low metal-to-protein ratio, do not give crystalline material. In the soaking experiment, Zeise's anion leads to simultaneous platination of His68, Met1 and Lys6. Present and previous results of co-crystallization experiments performed with zinc(II) and other group-12 metal ions allow to reach a comprehensive understanding of the metal-ion binding properties of hUb with His68 as main anchoring site, followed by Met1 and Glu18. The amount of metal ion, with respect to that of the protein, appears to be a relevant parameter influencing the crystal packing.