Fig 1.
Views of the tripartite assembly of an efflux pump.
A: The schematic reconstitution complex supported by microscopy images on the MexAB-OprM in nanodiscs and by the chimeric AcrAB-TolC-AcrZ complex [9]. The oligomerization state is 3:6:3 for the three components, respectively. The RND part uses the proton motive force and is responsible of the active counter transport of the drug. B: The overall trimer assembly of OprN (the OMF component -in red at left- of the MexEF-OprN assembly). C: Topology of secondary structures of the OprN monomer with β-strands S1 to S4 and α-helices H1 to H7.
Table 1.
Data recording statistics and refinements (T = 100 K) for the OprN-wt and the OprN-Xe complex.
Both are isomorphous, in the tetragonal I4 space group (Data from the highest resolution bin are in parentheses).
Fig 2.
A: The anomalous map—in black tracing and contour at 3.5 σ.—superimposed to the Cα tracing of the OprN structure. The three S1 to S3 sites are labelled. On top of the structure, outside of the β-barrel, four more xenon atoms are present around the β-barrel domain, in a fully hydrophobic environment. B: (view from the extra cellular side) The two inter and intra-subunit S2 and S3 sites in the helical α-domain (xenon as red spheres) viewed along the central channel of the trimer. C: (view from the periplasmic side) The M1 site locked by the three equivalent Leu 405, viewed along the central channel of OprN-Xe structure at the periplasmic entrance. The S1 secondary sites are located at the same level of the main M1 site in a plane perpendicular to the tunnel. S1 xenon atoms are in red, M1 xenon in lime green. The three monomers building the cylindrical scaffold are in three different colours.
Fig 3.
A: Details of the xenon's cavity M1 viewed along the tunnel. The cavity (in mesh) is built by the three hydrophobic Leu 405 side chains on one edge, and the circular salt bridge system [.. -> Asp 409a—Arg 412b → Asp 409b—Arg 412c → Asp 409c—Arg 412a →..] on the other. Hydrophobic contacts to Leu are in brown, hydrogen bonds between Glu and Arg are in green. B: Residues building the site S1, at the same level as M1. Note that the hydrophobic contacts towards polar residues like Gln or Arg, displace the polar part of their side chains as to keep the overall hydrophobic character of the cavity. Xenon atoms are represented as spheres with arbitrary radius.
Fig 4.
A: The huge central cavity of OprN constricted at both ends, towards the periplasm (bottom), and the exterior of the cell (top). B: At the same scale, the cavity's profile as seen by the ChExVis program [35] with the two constricted regions pointed by the two red arrows. The gradient profile of the cavity is given as the standard Kyte & Doolittle scale of polar/hydrophobic amino-acids (color code is blue to red for increasing hydropathy). C: Non-bonded energy calculated for a xenon atom rolling along the central tunnel of OprN, superposed to the same calculations with OprM and TolC structures (3d5K and 1ek9 from PDB). OprN, OprM, and TolC traces are in blue, orange and lime colors. Energies are calculated using the Lennard-Jones parameters from the CHARMM22 forcefield [34].