Structural Basis of Transcriptional Coactivator PC4 Binding to a Platinum Crosslinked Double-Stranded Oligonucleotide

Abstract We previously discovered that human nuclear cofactor PC4 selectively binds to a double-stranded oligodeoxynucleotide (dsODN) crosslinked by a trans -platinum anticancer complex, trans -[PtCl 2 (NH 3 )(thiazole)] ( trans- PtTz), which was further demonstrated to reduce its cytotoxicity by mediating DNA repair, though the molecular mechanism was unclear. In this work, we developed an amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) coupled to online peptic digestion to dissect interaction interface and binding sites between PC4 and a trans -PtTz crosslinked 15-mer dsODN ( trans -PtTz- III ). Using online HDX-MS, we identified a 1:1 binding stoichiometry and key involvement of the β3–β5 sheets (K80–Q109) in recognition. Molecular dynamic simulations suggest a 2:2 binding mode and the C-terminal helices slightly loosen, with Arg86 being critical for the recognition and interaction between PC4 and trans -PtTz- III . More importantly, site-directed mutation of Arg86 weakened the binding of PC4 to trans -PtTz- III in vitro and promoted the cytotoxicity of trans -PtTz against A549 cells. This work profiles the detailed interaction mechanism of PC4 with trans -PtTz damaged dsODN, and provides a new paradigm for the further research of the cellular response to platinum induced DNA damage.