Room-temperature X-ray diffraction studies of cisplatin and carboplatin binding to His15 of HEWL after prolonged chemical exposure

Abstract

The anticancer complexes cisplatin and carboplatin are known to bind to both the Nδ and the Nℇ atoms of His15 of hen egg-white lysozyme (HEWL) in the presence of dimethyl sulfoxide (DMSO). However, neither binds in aqueous media after 4 d of crystallization and crystal growth, suggesting that DMSO facilitates cisplatin/carboplatin binding to the N atoms of His15 by an unknown mechanism. Crystals of HEWL cocrystallized with cisplatin in both aqueous and DMSO media, of HEWL cocrystallized with carboplatin in DMSO medium and of HEWL cocrystallized with cisplatin and N-acetylglucosamine (NAG) in DMSO medium were stored for between seven and 15 months. X-ray diffraction studies of these crystals were carried out on a Bruker APEX II home-source diffractometer at room temperature. Room-temperature X-ray diffraction data collection removed the need for cryoprotectants to be used, ruling out any effect that the cryoprotectants might have had on binding to the protein. Both cisplatin and carboplatin still bind to both the Nδ and Nℇ atoms of His15 in DMSO media as expected, but more detail for the cyclobutanedicarboxylate (CBDC) moiety of carboplatin was observed at the Nℇ binding site. However, two molecules of cisplatin were now observed to be bound to His15 in aqueous conditions. The platinum peak positions were identified using anomalous difference electron-density maps as a cross-check with Fo-Fc OMIT electron-density maps. The occupancies of each binding site were calculated using SHELXTL. These results show that over time cisplatin binds to both N atoms of His15 of HEWL in aqueous media, whereas this binding is speeded up in the presence of DMSO. The implication of cisplatin binding to proteins after a prolonged period of time is an important consideration for the length of treatment in patients who are given cisplatin.

Safeguarding Structural Data Repositories against Bad Apples

“…One of the authors of this paper (JRH) received a valuable critique of a group of crystal structures from the other authors of this paper (Shabalin et al, 2015), to which a response was provided (Tanley et al, 2015). This back-and-forth process is leading to improvements in the associated PDB files and showcases the advantages of the availability of all the data (including raw diffraction images, processed structure factor amplitudes, and the final derived coordinates) underpinning the publications (Tanley et al, 2012) and the raw data (doi: 10.15127/1.215887 https://www.escholar.manchester.ac.uk/uk-ac-man-scw:215887). More widely, the fraction of questionable deposits, especially of protein-ligand complexes that are most important for drug discovery studies, is not decreasing with time.…”

Crystallography and chemistry should always go together: a cautionary tale of protein complexes with cisplatin and carboplatin

“…Whereas the vast majority of structures represent complexes with the tetragonal or triclinic forms of hen eggwhite lysozyme (HEWL; Casini et al, 2007;Tanley, Schreurs, Kroon-Batenburg & Helliwell, 2012b;Tanley, Schreurs, Kroon-Batenburg, Meredith et al, 2012;Tanley et al, 2014;Tanley & Helliwell, 2014a), data are also available for complexes with bovine Cu,Zn superoxide dismutase (SOD; Calderone et al, 2006;Banci et al, 2012), the human copper-transport protein ATOX1 (Boal & Rosenzweig, 2009), bovine pancreatic RNase A (Messori & Merlino, 2014) and Plasmodium falciparum glutaredoxin 1 (PfGrx1; unpublished work). All available structures of carboplatin involve complexes with the tetragonal, monoclinic or trigonal forms of HEWL (Tanley, Schreurs, Kroon-Batenburg & Helliwell, 2012a;Tanley, Schreurs, Kroon-Batenburg, Meredith et al, 2012;Tanley et al, 2014;Tanley & Helliwell, 2014b).…”

Structural dynamics of cisplatin binding to histidine in a protein

“…Cisplatin and carboplatin binding to hen egg white lysozyme (HEWL) as a model protein has been extensively studied using X-ray crystallography 15–21 Using the standard tetragonal HEWL crystallisation conditions (NaCl and NaAc) with the addition of dimethyl sulfoxide (DMSO) and cisplatin/carboplatin, the platinum anti-cancer agents bound to the Nδ and Nε atoms of His-15, 16–19 and even without DMSO i.e. in aqueous medium but requires prolonged chemical exposure 17 . A plausible role of the chloride ions is to extract the hydrogens from the histidine to create an imidazolyl ion.…”