Fig. 4: Binding site identification of Metro and Metro-P3.
a, The binding site identification workflow using an isotopically labelled desthiobiotin azide tags proteomic workflow42. b, The binding site identification results of Metro-P3-labelled H. pylori proteome. AA, amino acid residue; Cys, cysteine; tag, heavy/light isoDTB tag. c, The gel-based labelling of recombinant HpTpx wild-type or mutant (C60, C94A, C60A-C94A) HpTpx in E. coli. Top: fluorescence gel. Bottom: Coomassie gel as loading control. Data are shown from one experimental replicate. d, The co-crystal structure of HpTpx C94A mutant (HpTpx_CRA) with Metro* at 1.75 Å resolution (top) (PDB ID 9F64) and with Metro-P3* at 1.95 Å resolution (bottom) (PDB ID 9F65). Top: a ribbon diagram of the HpTpx_CRA mutant with Metro* (C atoms in purple). Cp and CRA are depicted in black and pink. The inhibitor is shown in a balls-and-sticks representation. Right: a close-up view of the active site (H bonds indicated by black dots; VdW, van der Waals). The 2Fo–Fc electron density map (grey mesh, contoured to confidence interval 1.0 σ) reveals full occupancy for Metro*. Bottom: a ribbon structure of HpTpx_CRA bound to Metro-P3* (P3*, carbon atoms in cyan). The colour coding and orientation are according to the top. e, The superposition of HpTpxred (orange, PDB ID 9F5V) with HpTpx_CRA:Metro* (purple) and HpTpx_CRA:Metro-P3* (cyan). The covalent binding of Metro* results in notable structural rearrangements in the Cp region. *Reduced amine form of nitro-prodrugs. DTB, desthiobiotin azide.
