Metallodrug-Protein Interactions at Physiological Temperature: Structural Insights from X-ray Crystallography at 37 °C

Protein metalation is a fundamental biological process through which proteins interact with metal ions or complexes, leading to the formation of stable metal–protein adducts. Understanding protein metalation under physiologically relevant conditions is essential for gaining deeper insights into the mechanisms of action of metallodrugs. Temperature is a key factor that can affect metal coordination geometry, ligand exchange processes, and protein conformational flexibility. Therefore, structural studies performed at physiological temperature can provide a more representative picture of metal–protein interactions in biological systems.

We have developed an experimental protocol for the growth of crystals of adducts formed upon reaction of metallodrugs with proteins at 37 °C and for the collection of X-ray diffraction data at the same temperature. X-ray structures of the adducts formed by the model protein hen egg white lysozyme (HEWL) with bis(acetylacetonato)oxidovanadium(IV), [VᴵⱽO(acac)₂], and dirhodium tetraacetate, [Rh₂(μ-O₂CCH₃)₄] were obtained and solved. Then, the resulting structures were compared with those previously determined at cryogenic temperature (100 K). At 37 °C, the reaction of [VᴵⱽO(acac)₂] with HEWL results in the formation of polyoxidovanadate (POV) clusters whose structural features differ markedly from those observed at 100 K. In contrast, the structure of the [Rh₂(μ-O₂CCH₃)₄]/HEWL adduct at 37 °C remains largely comparable to that determined at 100 K, with differences mainly involving metal occupancy and local residue mobility.

Overall, these findings highlight the significant influence of temperature on metal coordination and speciation in metal–protein systems and demonstrate the importance of performing structural investigations at physiological temperature to obtain a more realistic description of metallodrug–protein interactions and their potential biological behaviour.