The C-terminus of the multi-drug efflux pump EmrE prevents proton leak by gating transport

Reviewer #1 (Public review):

Summary:

Work by Brosseau et. al. combines NMR, biochemical assays, and MD simulations to characterize the influence of the C-terminal tail of EmrE, a model multi-drug efflux pump, on proton leak. The authors compare the WT pump to a C-terminal tail deletion, delta_107, finding that the mutant has increased proton leak in proteoliposome assays, shifted pH dependence with a new titratable residue, faster-alternating access at high pH values, and reduced growth, consistent with proton leak of the PMF.

Strengths:

The work combines thorough experimental analysis of structural, dynamic, and electrochemical properties of the mutant relative to WT proteins. The computational work is well aligned in vision and analysis. Although all questions are not answered, the authors lay out a logical exploration of the possible explanations.

Weaknesses:

There are a few analyses that are missing and important data left out. For example, the relative rate of drug efflux of the mutant should be reported to justify the focus on proton leak. Additionally, the correlation between structural interactions should be directly analyzed and the mutant PMF also analyzed to justify the claims based on hydration alone. Some aspects of the increased dynamics at high pH due to a potential salt bridge are not clear.

Reviewer #2 (Public review):

Summary:

This manuscript explores the role of the C-terminal tail of EmrE in controlling uncoupled proton flux. Leakage occurs in the wild-type transporter under certain conditions but is amplified in the C-terminal truncation mutant D107. The authors use an impressive combination of growth assays, transport assays, NMR on WT and mutants with and without key substrates, classical MD, and reactive MD to address this problem. Overall, I think that the claims are well supported by the data, but I am most concerned about the reproducibility of the MD data, initial structures used for simulations, and the stochasticity of the water wire formation. These can all be addressed in a revision with more simulations as I point out below. I want to point out that the discussion was very nicely written, and I enjoyed reading the summary of the data and the connection to other studies very much.

Strengths:

The Henzler-Wildman lab is at the forefront of using quantitative experiments to probe the peculiarities in transporter biophysics, and the MD work from the Voth lab complements the experiments quite well. The sheer number of different types of experimental and computational approaches performed here is impressive.

Weaknesses:

The primary weaknesses are related to the reproducibility of the MD results with regard to the formation of water wires in the WT and truncation mutant. This could be resolved with simulations starting from structures built using very different loops and C-terminal tails.

The water wire gates identified in the MD should be tested experimentally with site-directed mutagenesis to determine if those residues do impact leak.