Solid-state NMR Reveals Asymmetric ATP hydrolysis in the Multidrug ABC Transporter BmrA

Abstract

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in pre-hydrolytic, transition and post-hydrolytic states, using a combination of mutants and ATP analogues. The 31P spectra reveal that ATP binds strongly in the pre-hydrolytic state to both ATP-binding sites as inferred from the analysis of the non-hydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg2+:vanadate, while the second site is more ‘open’ allowing exchange with the nucleotides in the solvent. In the post-hydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31P-31P 2D correlation spectra, is observed for both sites. Revisiting the 13C spectra in light of these findings confirms the conformational non-equivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP hydrolysis step, but is then recovered in the post-hydrolytic ADP-bound state.