An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity

Abstract

Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.

Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism

“…5C), causing the cavity to remain open. This result fits well with the presence of a detergent molecule in the binding pocket of McjD (8) together with the very recent discovery of a lipid inside the structure of the Major Facilitator Superfamily protein LrmP (38). Altogether, this data highlights the hydrophobicity of the drug-binding pocket as the main driving force for the closing movement, independently of any ATP hydrolysis.…”

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

“…8D). The mechanism of LmrP-dependent Hoechst uptake might relate to published findings 27,28 that Hoechst binding to purified LmrP at pH 8.0 stabilises an outwardfacing conformation rather than the inward-facing conformation. Together with our results, this suggests that Hoechst initially binds to LmrP at the outside of the membrane from where transport is initiated, leading to LmrP-mediated Hoechst uptake.…”

“…This phenomenon reflects the different mechanisms of drug binding in the interior chamber of LmrP, some of which alter the availability of catalytic carboxylates for proton interactions 24,25 . Hoechst transport and binding have also been used to characterise drug interactions in LmrP 6,23,26 , to define steps in the transport cycle in structural analyses 27 and, most recently, to trap the protein in a conformation that could be crystallised, thus allowing the determination of the three-dimensional protein structure 28 .…”

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug ABC transporter BmrCD reveals modes of asymmetric drug binding

“…All simulations were carried out with NAMD 2.12 51 using the CHARMM36 force field 52,53 periodic boundary conditions, constant temperature (298 K) and semi-isotropic pressure (1 atm), and an integration time step of 2 fs. Force-field parameters for Hoechst-33342 were those developed in a previous study 30 . Long-range electrostatic interactions were calculated using PME, with a real-space cut-off of 12 Å; van der Waals interactions were computed with a Lennard-Jones potential cut-off at 12 Å with a smooth switching function taking effect at 10 Å.…”

“…Lastly, it is worth noting that these interaction patterns recapitulate what is observed in high-resolution crystal structures of other biomolecules in complex with Hoechst. In the In the outward-open structure of the MFS-family multi-drug transporter LmrP 30 , a Hoechst molecule is stabilized through ion-pairs between two acidic side chains and the piperazine and benzimidalole groups, albeit in a different orientation nearly perpendicular to the plane of the transmembrane. The ligand is however only in contact with one of the transmembrane domains, like in our structure of inward-open BmrCD.…”