Figure 3. Nucleotide states in the sandwiched NBD dimer and opening of the intracellular gate.

a, Different nucleotide states were observed at the canonical nucleotide-binding site in the conformations with dimerized NBDs from TmrA^EQB_ATP-ADP, TmrAB_ATP-Vi, and TmrAB_turnover (highest resolution map presented). In all cases, the nucleotide-binding pocket is aligned by the conserved Walker A/B motives and the signature motif. Mg²⁺ and water molecules are shown as green and red spheres, respectively. TmrA is blue, TmrB yellow. b, The non-canonical site is occupied by ATP in all examples, as hydrolysis cannot proceed. OF^open from TmrA^EQB_ATP-ADP is shown as an example. In the non-canonical site, the sugar moiety of the bound nucleotide interacts with R351^TmrB and E501^TmrA, while a tyrosine residue (Y362^TmrA) makes π-π stacking contacts with the purine base in the canonical site. c, The stoichiometric occlusion of ADP and ATP was validated by thin-layer chromatography upon vanadate-trapping of TmrAB^WT (mean ± SD from three experiments). d, MD snapshot showing two possible exit pathways for inorganic phosphate (P_i) after hydrolysis in a cut through the NBD dimer. P_i can escape through hydrated channels directed towards the C-terminal helices (bottom) and the coupling helix (top). The OF^occluded conformation of TmrAB_ATP-Vi was simulated with bound ADP and P_i (HPO₄^2–). e, The NBD dimer, TM3, and TM4 show the largest differences between OF^occluded and UR^asym* conformations. Red color and wide tubes indicate large Cα distances. The distances were averaged over two OF^occluded (from TmrA^EQB_ATP-ADP and TmrAB_ATP-Vi) and the UR^asym* conformation in a backbone superposition excluding the N-terminal elbow and C-terminal helices. The TM3 and TM4 motions from OF to UR^asym* progressively separate the intracellular gate. f, After P_i release, the intracellular gate lined by TM4 and TM3 of each subunit is unlocked, forming the asymmetric turnover state UR^asym*. OF^occluded is transparent.