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. 2019 Apr 29:2:149.
doi: 10.1038/s42003-019-0390-x. eCollection 2019.

Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis

Denis Lacabanne #  1 , Cédric Orelle #  1 , Lauriane Lecoq  1 , Britta Kunert  1 , Claire Chuilon  1 , Thomas Wiegand  2 , Stéphanie Ravaud  1 , Jean-Michel Jault  1 , Beat H Meier  2 , Anja Böckmann  1
Affiliations

Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis

Denis Lacabanne et al. Commun Biol. .

Abstract

ATP-binding-cassette (ABC) transporters are molecular pumps that translocate molecules across the cell membrane by switching between inward-facing and outward-facing states. To obtain a detailed understanding of their mechanism remains a challenge to structural biology, as these proteins are notoriously difficult to study at the molecular level in their active, membrane-inserted form. Here we use solid-state NMR to investigate the multidrug ABC transporter BmrA reconstituted in lipids. We identify the chemical-shift differences between the inward-facing, and outward-facing state induced by ATP:Mg2+:Vi addition. Analysis of an X-loop mutant, for which we show that ATPase and transport activities are uncoupled, reveals an incomplete transition to the outward-facing state upon ATP:Mg2+:Vi addition, notably lacking the decrease in dynamics of a defined set of residues observed in wild-type BmrA. This suggests that this stiffening is required for an efficient transmission of the conformational changes to allow proper transport of substrate by the pump.

Keywords: Bacteria; Solid-state NMR.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
ABC transporter export cycle and samples studied. a Putative export cycle of an ABC exporter adapted from Grossmann et al. and Parcej et al.. A sketched model of the BmrA homodimer is given in gray, ATP is indicated in red, and ADP in yellow. Bound nucleotides in undetermined hydrolysis states are shown in gray. b Mimics of different BmrA states investigated, as desribed in the text. c BmrA homology model based on Sav1866 (pdb 2hyd2), the two mononers are shown in light gray and dark gray colors. Mutated residues are highligthed, with E474 from the X-loop motif (470-TEVGERG-476) in green, residue K380 from the Walker A motif (374-GPSGGKT-381) in magenta, and residue E504 from Walker B (496-ILMLDE-504) in dark yellow. The ABC signature (477-LSGGQ-483, in blue) and the H-loop (532-AHR-536, in cyan) motifs are also presented. d View of the NBDs of BmrA modeled on Sav1866 (pdb 2hyd). A362, A371, A505, and A534 (as discussed in the text) are shown in black spheres, with the Mg2+ ions represented in light green sphere
Fig. 2
Fig. 2
Spectral differences between investigated protein states (for additional regions see Supplementary Fig. 3A, 4A and 6B). a wt:apo (IF) and wt:ADP:Mg:Vi (OF) states display different spectral fingerprints. 2D DARR extracts showing Ala region peaks for wt:apo (IF) (red) and wt:ADP:Mg:Vi (OF) (cyan, spectrum from Wiegand et al.) states. Analyzed peaks are marked in all panels by crosses. Signals only observed in the wt:ADP:Mg:Vi (OF) state are highlighted by red circles (middle panel). b PRE attenuates signals near the Mn binding site. Comparison between spectra of wt:ADP:Mg:Vi and wt:ADP:Mn:Vi (OF) (left and middle panels, spectrum from Wiegand et al.). Peaks erased by PRE are highlighted by purple circles (middle panel). Comparison of wt:apo (IF) and wt:ADP:Mn:Vi (OF) (right panel), red/purple circles highlight the signals observed in neither state. c Mimics of the prehydrolytic and transition states display similar conformations. Same spectral regions comparing the E504A:ATP:Mg (OF) state (dark blue) to the wt:ADP:Mg:Vi (OF) state in cyan; the spectra are, with exception of the modifications due to the mutation highlighted by circles, virtually identical
Fig. 3
Fig. 3
Transport and ATPase activity of BmrA mutants. a ATP-dependent transport of doxorubicin measured using inverted E. coli membrane vesicles. After addition of 10 µM doxorubicin (120 s), 2 mM of Mg2+ were added to initiate transport (indicated by black arrow). b Transport activities derived from initial rate fluorescence decays measured for BmrA wild-type and mutant forms, normalized to the rate measured in the wild-type form. c ATPase activity of BmrA wild-type and mutant forms purified and reconstituted in E. coli lipids with a lipid/protein ratio of 20. Each experiment was conducted three times and error bars indicate the standard deviation
Fig. 4
Fig. 4
The E474R:ADP:Mg:Vi mutant shares features with both the wt:apo (IF) and wt:ADP:Mg:Vi (OF). a extract of 2D DARR spectra of the wt:ADP:Mg:Vi (OF) (for larger extracts see Supplementary Fig. 12). b E474R:ADP:Mg:Vi, with brown circles representing peaks which are absent when compared with wt:ADP:Mg:Vi (OF) (Fig. 4a). c Overlay of the spectra shown in (a) and (b). d Overlay of the extracts shown in (b) with the wild-type protein inward-facing state. Brown-red circles highlight the signals which are only observed in wt:ADP:Mg:Vi (OF), as shown in (a). e Differences between the CSPE474R:ADP:Mg:Vi/wt:ADP:Mg:Vi (OF) and the CSPE474R:ADP:Mg:Vi/wt:apo (IF). Red positive bars indicate that the CSPE474R:ADP:Mg:Vi/wt:apo (IF) is smaller than the CSP E474R:ADP:Mg:Vi/wt:ADP:Mg:Vi (OF), and negative cyan bars indicate that the CSPE474R:ADP:Mg:Vi/wt:ADP:Mg:Vi (OF) is smaller than the CSP CSPE474R:ADP:Mg:Vi/wt:apo (IF). “α” indicates that the peak is located in a region where α-helical chemical shifts are located
Fig. 5
Fig. 5
Summary of our findings (highlighted in red). a BmrA in its inward-facing state with a subset of residues showing dynamics on the microsecond time scale, which are mainly located within 15 Å of the ATP:Mg-binding site. b, c BmrA in its outward-facing state, in which a set of residues stiffens, and conformational changes occur around the ATP-binding site, but also remotely in an allosteric manner to allow drug transport. d In the presence of ATP:Mg:Vi, the E474R mutant makes an incomplete conformational and dynamic transition, with the NBDs engaged in and ADP:Mg:Vi bound state, but with residues remaining flexible, and only partially observed CSPs
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