The binding of platinum hexahalides (Cl, Br and I) to hen egg-white lysozyme and the chemical transformation of the PtI6 octahedral complex to a PtI3 moiety bound to His15

The platinum hexahalides have an octahedral arrangement of six halogen atoms bound to a Pt centre, thus having an octahedral shape that could prove to be useful in interpreting poor electron-density maps. In a detailed characterization, PtI6 chemically transformed to a square-planar PtI3 complex bound to the Nδ atom of His15 of HEWL was also observed, which was not observed for PtBr6 or PtCl6.


S1. HEWL+K 2 PtBr 6
After soaking HEWL crystals with K 2 PtBr 6 for 24hrs, an octahedral PtBr 6 molecule is bound to site 1 (using the naming convention of Helliwell et al 2010) which is at a special position between two Arg-14 residues in symmetry related molecules with a two-fold axis passing through the platinum atom and two bromines ( Figure S1). Also, an octahedral complex is bound in site two near to Ser-86, Lys-1 and Gln-41 of chain A in a crevice next to Pro-79, Asn-65 and Asn-74 in a symmetry related molecule ( Figure S2), similar to the 3hr soak study (Helliwell et al, 2010). However, no PtBr 3 moiety is bound to the Nδ atom of His-15.
A large anomalous difference electron density peak of 8σ is observed 2.4Å away from the sulphur atom of Cys-6, which is disulphide bonded to Cys-127. A platinum atom is assumed bound due to the distance from the S atom. Binding of platinum to disulphide bonded Cys residues has been previously described by Moreno-Gordaliza et al, (2009;2010) using mass spectrometry to study the interaction of cisplatin with insulin.

Figure S1
PtBr 6 binding in a special position between two Arg-14 residues in symmetry related molecules. The 2Fo-Fc electron density map (blue) and the anomalous difference electron density map (orange) are shown. The platinum atom is in grey and bromine atoms in red.

S2. HEWL+K 2 PtCl 6
After soaking HEWL crystals with K 2 PtCl 6 for 24hrs, an octahedral PtCl 6 molecule is seen bound to site 1, i.e. a special position between two Arg-14 residues in symmetry related molecules with a twofold axis passing through the platinum atom and three chlorines ( Figure S3). Also, an octahedral complex is bound in site two near Pro-79, Asn-65 and Asn-74 of chain A in a crevice next to Ser-86, Lys-1 and Gln-41 in a symmetry related molecule ( Figure S2), similar to the 10min soak study (Sun et al, 2002). However, no PtCl 3 moiety is seen bound to the Nδ atom of His-15.  Supporting information, sup-5

S3. Refinement of heavy atom occupancies and B factors
Refinement of heavy atom occupancies and B factors; given the different mathematical functional forms of these two parameters, provided the diffraction resolution is adequate, these two parameters can be refined simultaneously. Furthermore monitoring of how physically sensible the values obtained are form a check on the adequacy of the diffraction resolution and data quality.

S4. Other remarks on binding sites
In Helliwell et al (2010) a minor occupancy PtBr 6 site ('site 3') at the 20% level was seen and specifically whose occupancy did not increase with soak time up to 3 hours. In the 24hr soaking experiment reported in this new study this site 3 is not occupied at all for any of the Pt hexahalide cases. In another case, the Cys-6-Cys-127 disulphide bond in the HEWL K 2 PtBr 6 3hr study shows anomalous difference electron density, proximal to the nearest sulphur atom and therefore possibly a platinum atom. In this 24hr K 2 PtBr 6 soaking experiment, there is some similar density but even less clear than the 3hr soak study. Previous mass spectrometry studies of cisplatin binding to insulin have reported cisplatin binding to Cys residues partaking in disulphide bond formation (Moreno-Gordaliza et al, 2009;2010). The reason for these differences between the 24hr and 3hr K 2 PtBr 6 soaking studies with HEWL are not known.
Besides soaking times being different, the Helliwell et al, 2010 studies used glycerol as the cryoprotectant rather than paratone as reported in these current studies. However this particular difference seems unlikely to be relevant. In both the PtCl 6 and PtI 6 cases, no anomalous difference electron density is observed near the Cys-6-Cys-127 disulphide bond.
The occupancy values for the Pt atom in PtBr6 (Table S3) at sites one and two are 46% and 54%.
These have stayed at a similar occupancy as the 3hr study (Helliwell et al, 2010), thus increasing the soaking times from 3hr to 24hrs has not improved the binding occupancy at these two sites.