Zooming in on the conformational details of a membrane efflux pump – BmrA-NMX

To answer these questions, we will focus on the bacterial ABC transporter BmrA, which is structurally and functionally close to ABCB1, the human P-gp (mainly responsible for drug resistance in humans38), and will study its conformations and interactions with drugs using the combination of solid-state NMR (N), H/D exchange coupled with mass spectrometry (M) and x-ray crystallography (X). The partners will aim at combining their key competences in a concerted manner to isolate and characterize new conformational intermediates, in order to draw a most complete picture of the processes involved in drug binding and export.

- Development of proton detection NMR techniques
-Development of NMR chemical shift fingerprinting methods in large multidomain proteins (using as a model the DnaB helicase)
-Sample preparations of reversely labeled BmrA was established reinserted into lipid membranes.
-Exploring alternative lipid reconstitution techniques (GRecon).
-The protein was prepared in different functional states corresponding to the open, closed, and drug-bound forms.
-Paramagnetic analogues of the protein were prepared.
-Spectra of the transporter were analyzed
-Different mutant forms were established and tested for activity
Establish proton detection of the samples in order to extend assignments; analyze data from open/closed/drug bound states

We have created new mutants in the so-called ‘X-loop’ and ‘Q-loop’ of BmrA based on NMR spectra suggesting the very unusual chemical shift of either a Glu or Gln residue (surrounded by a Gly and Arg residues (sequence G(E/Q)R or R(E/Q)G). Crippled transport activities have been measured for the mutant and purification of the proteins suggest that the ATPase activities are unaltered. Therefore these mutants are affected in the coupling between ATPase and transport activities. Conditions of limited proteolysis have been optimized to support the existence of a different conformation in the presence of transported drugs. They have been validated at the level of the membrane fractions and after reconstitution into proteoliposomes.
We have focused on BmrA crystallization, so far obtained in DDM-cholate at a resolution of 6 A. in 2016, we have reproduced the condition and we are testing other detergents, starting with LMNG replacing DDM. We are quantifying those detergents by a method we have set up using MALDI tof MS (submitted).
Many crystallization trials have been performed in lipidic mesophases on the High throughput membrane protein crystallization of the ISBG in Grenoble. The tests have been conducted on the wild type and on inactive mutant of BmrA in various detergent and concentration conditions. A few of them lead to crystalline objects that could be seen under UV light suggesting they are made of protein. They have been tested at the ESRF on a microfocus beamline but did not show any X-ray diffraction. Optimization trials are currently on going.

1. T. Wiegand, C. Gardiennet, R. Cadalbert, D. Lacabanne, B. Kunert, L. Terradot, A. Böckmann, and B. H. Meier, “Variability and conservation of structural domains in divide-and-conquer approaches.,” J. Biomol. NMR, pp. 1–8, May 2016.
2. C. Gardiennet, T. Wiegand, A. Bazin, R. Cadalbert, B. Kunert, D. Lacabanne, I. Gutsche, L. Terradot, B. H. Meier, and A. Böckmann, “Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori,” J. Biomol. NMR, vol. 64, no. 3, pp. 189–195, Mar. 2016.
3. T. Wiegand, C. Gardiennet, F. Ravotti, A. Bazin, B. Kunert, D. Lacabanne, R. Cadalbert, P. Güntert, L. Terradot, A. Böckmann, and B. H. Meier, “Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase,” Biomol NMR Assign, pp. 1–11, 2015.
4. S. Penzel, A. A. Smith, V. Agarwal, A. Hunkeler, M.-L. Org, A. Samoson, A. Böckmann, M. Ernst, and B. H. Meier, “Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods.,” J. Biomol. NMR, vol. 63, no. 2, pp. 165–186, Oct. 2015.
5. A. Böckmann, M. Ernst, and B. H. Meier, “Spinning proteins, the faster, the better?,” Journal of Magnetic Resonance, vol. 253, no. C, pp. 71–79, Apr. 2015.

We will seek seek in this BmrA-NMX project to reveal structure features of the different conformational states of BmrA, an ABC efflux pump, using N for NMR, M for Mass spectrometry, and X for X-ray crystallography methods. BmrA is, other members of ABC transporters family, able to transport a wide variety of drugs. Multidrug resistance due to efflux pumps has strong implication in medicine because it is found from humans to bacteria.
We will use state-of-the art biochemical studies to identify and characterize different conformational states, using wild-type and mutant forms, and drug-bound forms. Drug binding and transport abilities of the corresponding states of BmrA will be quantified and transposed to crystallogenesis, mass spectrometry and solid-state NMR experiments. These approaches will be used to gain insight into the structural features of the membrane pump. They will deliver complementary structural parameters, such as the overall 3D structure, detailed information on conformational changes as a function of state, and stability and accessibility of secondary structure elements. Sample preparations of the different forms will be established using feed back from the biochemical studies, and crystalline, as well as membrane-bound, drug-bound and mutant forms will be made for the different studies.
The unique combination of structural techniques is expected to obtain a comprehensive picture of the processes involved in drug export. Such knowledge will be an important steppingstone towards deciphering the molecular mechanism underlying drug export.