Deciphering of interactions between platinated DNA and HMGB1 by hydrogen/deuterium exchange mass spectrometryElectronic supplementary information (ESI) available: The supplementary Table S1 and Fig. S1-S9. See DOI: 10.1039/c7dt00275k

A high mobility group box 1 (HMGB1) protein has been reported to recognize both 1,2-intrastrand crosslinked DNA by cisplatin (1,2- cis -Pt-DNA) and monofunctional platinated DNA using trans -[PtCl 2 (NH 3 )(thiazole)] (1- trans -PtTz-DNA). However, the molecular basis of recognition between the trans -PtTz-DNA and HMGB1 remains unclear. In the present work, we described a hydrogen/deuterium exchange mass spectrometry (HDX-MS) method in combination with docking simulation to decipher the interactions of platinated DNA with domain A of HMGB1. The global deuterium uptake results indicated that 1- trans -PtTz-DNA bound to HMGB1a slightly tighter than the 1,2- cis -Pt-DNA. The local deuterium uptake at the peptide level revealed that the helices I and II, and loop 1 of HMGB1a were involved in the interactions with both platinated DNA adducts. However, docking simulation disclosed different H-bonding networks and distinct DNA-backbone orientations in the two Pt-DNA-HMGB1a complexes. Moreover, the Phe37 residue of HMGB1a was shown to play a key role in the recognition between HMGB1a and the platinated DNAs. In the cis -Pt-DNA-HMGB1a complex, the phenyl ring of Phe37 intercalates into a hydrophobic notch created by the two platinated guanines, while in the trans -PtTz-DNA-HMGB1a complex the phenyl ring appears to intercalate into a hydrophobic crevice formed by the platinated guanine and the opposite adenine in the complementary strand, forming a penta-layer π-π stacking associated with the adjacent thymine and the thiazole ligand. This work demonstrates that HDX-MS associated with docking simulation is a powerful tool to elucidate the interactions between platinated DNAs and proteins. The Phe37 residue in HMGB1a forms multi-layer π-π stacking, playing a crucial role in the interaction of HMGB1a with mono-transplatinated DNA.