Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

Abstract

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. MFS proteins are known to adopt different conformational states, yet details on the interconversion rates are crucially needed to understand or target their transport mechanism. Here, we studied the proton/multidrug antiporter LmrP as a model system for antibiotic resistance development in bacteria. The conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, yet the role of the transported substrate in these transitions has been puzzling. To measure LmrP structure in real-time, we performed solution-based single-molecule Förster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein interconverted between two structures at low rate (>>10 ms dwell time) at the cytosolic side while it interconverts dynamically BRIEF SUMMARYWe studied the conformational cycle of LmrP, a model for multidrug efflux pumps, using single-molecule Förster resonance energy transfer (smFRET). By following changes in FRET signal between different sets of positions, we specifically investigated how substrate binding modulates structural conversions between inward-open and outward-open states. Using a newly developed probabilistic analysis for describing sequential interconversion kinetics, we show that the apo protein slowly interconverts between defined states at the cytosolic and at the extracellular sides. Binding of the model substrate Hoechst33342 leads to an increase in conformational interconversions at the intracellular side while the extracellular side shows a drastic decrease in conversion, indicating a kinetic uncoupling between both sides. Remarkably, binding of roxithromycin, while also increasing interconversion on the intracellular side, did not slow the extracellular conversions. This indicates that multidrug pumps have evolved substrate-dependent transport mechanisms than enable transport of structurally diverse collection of substrates.

Single‐Molecule FRET of Membrane Transport Proteins

“…[90][91][92][93][94][95][96] The impact of mutations on function can be monitored by ATPase assays [79,90,[97][98][99][100][101] or transport assays. [78,90,[105][106][107][108]93,94,96,97,100,[102][103][104] In transport assays, the protein is either overexpressed in a cell or incorporated into liposomes and the accumulation of fluorescent [78,97,105] or radiolabeled substrate [90,93,94,96,100,102,103,[106][107][108] in the compartment (cell or vesicle) is measured. Good examples for such careful controls are LmrP transporter variants whose activities were checked with the fluorescent ligand Hoechst, [105] or variants of the Glt Ph transporter in liposomes where the uptake of the radioactive substrate [ 3 H]-Asp was used to confirm activity.…”

“…[78,90,[105][106][107][108]93,94,96,97,100,[102][103][104] In transport assays, the protein is either overexpressed in a cell or incorporated into liposomes and the accumulation of fluorescent [78,97,105] or radiolabeled substrate [90,93,94,96,100,102,103,[106][107][108] in the compartment (cell or vesicle) is measured. Good examples for such careful controls are LmrP transporter variants whose activities were checked with the fluorescent ligand Hoechst, [105] or variants of the Glt Ph transporter in liposomes where the uptake of the radioactive substrate [ 3 H]-Asp was used to confirm activity. [108] An elegant tool to fully avoid direct protein modifications are smFRET sensors that selectively probe the presence of substrates in vesicles (Figure 3).…”

“…[121] Thus numerous smFRET studies have been performed using detergents [121][122][123] and provided important insights. [78,79,108,[91][92][93][94]99,102,104,105] Yet, studies of transporters in membrane environments, which better mimic native conditions, showed that lipids play important roles in maintaining their structural and functional integrity. [120,[124][125][126][127][128] For certain transporters such as Glt Ph , dynamics in micelles do not differ significantly from those found in a lipid bilayer.…”

“…[89,91,92,163] In its apo state, the multidrug efflux protein MdfA from E. coli and the multidrug efflux pump LmrP from Lactococcus lactis adopt inward-open and outward-open states in detergent solution. [92,105] The peptide transporter DtpA and the galactoside permease LacY from E. coli on the contrary almost exclusively populate inward-open states under these conditions. [89,91] Whereas substrate binding shifted the inward-open to an outward-open conformation in LacY, [91] no switch had been observed for DtpA.…”

“…[89] Similarly, for LmrP an "extra-open" state on the periplasmic side and an "inward-very-closed" state were observed in detergent. [105] Unfortunately, the role of these additional conformations is currently elusive. Interestingly, MFS transporters operate at different timescales.…”