Fig. 2

NQO1 (NAD(P)H dehydrogenase quinone 1) and GSTP1 (glutathione S-transferase Pi 1) are the major MNPC-binding proteins. a Affinity chromatography of cell-free extract from U87MG/EGFRvIII cells. Aliquots of fractions were separated by SDS-PAGE. Bands were visualized by silver staining. CE, crude extract. MNPC, protein eluted with 1 mM MNPC in PBS b Amino acid sequence of the 29 kDa and 25 kDa bands that were identified by MS/MS as NQO1 and GSTP1. c BIACore X-100 binding assay for NQO1 and GSTP1. The kinetic parameters KD were measured by Biacore evaluation software. d X-ray crystal structure of NQO1 in complex with MNPC (PDB ID 6LLC). Surface representation of NQO1 in complex with MNPC bound in the active pocket formed in the dimer interface. The benzene ring of MNPC (yellow) was sandwiched by Phe178 and FAD (Brown) by π–π stacking. e Key interactions between MNPC and NQO1, including hydrogen bond with His161 and hydrophobic interactions with Tyr126, Tyr128, M131, Phe236 from one monomer and Trp105, Phe106, Met154, Tyr155 from another monomer. f Interactions between MNPC and GSTP1 are generated by molecular docking. Key interactions between MNPC and GSTP1, including hydrogen bonds with Arg100, Tyr108 and GSH, π–π stacking with Phe8 and Tyr108