Biology subjects

Henzler-Wildman, K. A.

Publications and source records attributed to Henzler-Wildman, K. A..

10 recordsLinked to original sources

bioRxiv2025

Energetic and structural control of polyspecificity in a multidrug transporter

Multidrug efflux pumps are dynamic molecular machines that drive antibiotic resistance by harnessing ion gradients to export chemically diverse substrates. Despite their clinical importance, the molecular principles underlying multidrug promiscuity and energy efficiency remain poorly understood. Using multiparametric deep mutational scanning across eight substrates and two energy conditions, we deconvolute the contributions of substrate recognition, energetic coupling, and protein stability, providing an integrated, high-resolution view of multidrug transport. We find that substrate specificity arises from a distributed network of residues extending beyond the binding site, with mutations that reshape binding, coupling, conformational flexibility, and membrane interactions. Further, we apply a pH-based selection scheme to measure the effect of mutation on pH-dependent transport efficiency. By integrating these data, we reveal a fundamental relationship between efficiency and promiscuity: highly efficient variants exhibit broad substrate profiles, while inefficient variants are narrower. These findings establish a direct link between energy coupling and polyspecificity, uncovering the biochemical logic underlying multidrug transport.

biochemistry↗

bioRxiv2025

Backbone and Sidechain 1H, 15N and 13C Resonance Assignments of a Multidrug Efflux Membrane Protein using Solution and Solid-State NMR

EmrE is a bacterial membrane-embedded multidrug transporter that functions as an asymmetric homodimer. EmrE is implicated in antibiotic resistance, but is now known to confer either resistance or susceptibility depending on the identity of the small molecule substrate. Here, we report both solution- and solid-state NMR assignments of S64V-EmrE at pH 5.8, below the pKa of critical residues E14 and H110. This includes 1H, 15N, and 13C resonance assignments of the backbone, methyl groups (isoleucine, leucine, valine, threonine and alanine) from solution NMR experiments in bicelles, and backbone and side-chain assignments from solid-state NMR 13C-detected experiments in liposomes.

biophysics↗

bioRxiv2024

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

The model multi-drug efflux pump from Escherichia coli, EmrE, can perform multiple types of transport leading to different biological outcomes, conferring resistance to some drug substrates and enhancing susceptibility to others. While transporters have traditionally been classified as antiporters, symporters, or uniporters, there is growing recognition that some transporters may exhibit mixed modalities. This raises new questions about the regulation and mechanisms of these transporters. Here we show that the C-terminal tail of EmrE acts as a secondary gate, preventing proton leak in the absence of drug. Substrate binding unlocks this gate, allowing transport to proceed. Truncation of the C-terminal tail ({Delta}107-EmrE) leads to altered pH regulation of alternating access, an important kinetic step in the transport cycle, as measured by NMR. {Delta}107-EmrE has increased proton leak in proteoliposome assays and bacteria expressing this mutant have reduced growth. MD simulations of {Delta}107-EmrE show formation of a water wire from the open face of the transporter to the primary binding site in the core, facilitating proton leak. In WT-EmrE, the C-terminal tail forms specific interactions that block formation of the water wire. Together these data strongly support the C-terminus of EmrE acting as a secondary gate that regulates access to the primary binding site in the core of the transporter.

biophysics↗

bioRxiv2023

A Small Multidrug Resistance Transporter in Pseudomonas aeruginosa Confers Substrate-Specific Resistance or Susceptibility

Small Multidrug Resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the {Delta}pH component of the Proton Motive Force (PMF), reducing viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from P. aeruginosa, transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of E. coli expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and in vitro solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of P. aeruginosa strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting P. aeruginosa can compensate for the proton leak occurring through PAsmr. These phenotypic differences between P. aeruginosa and E. coli advance our understanding of underlying resistance mechanisms in P. aeruginosa and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens. IMPORTANCESmall multidrug resistance transporters are a class of efflux pumps found in many pathogens, but whose contributions to antibiotic resistance are not fully understood. We hypothesize that these transporters may confer not only resistance, but also susceptibility, by dissipating the proton-motive force. This means to use an SMR transporter as a target, it merely needs to be present (as opposed to being the primary resistance mechanism). Here, we test this hypothesis with an SMR transporter found in Pseudomonas aeruginosa and find that it can perform both antiport (conferring resistance) and substrate-gated proton leak. Proton leak is detrimental to growth in E. coli but not P. aeruginosa, suggesting that P. aeruginosa responds differently to or can altogether prevent {Delta}pH dissipation.

biochemistry↗

bioRxiv2022

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells and were recently divided into two subfamilies: specific toxic metabolite transporters and promiscuous drug exporters. These drug exporters are thought to function similarly to EmrE, the model system for this subfamily of SMR transporters. Studies of EmrE homologs indicate that they are able to confer resistance to EmrE substrates in E. coli and in their native organisms. Recent work from our lab showed that functional EmrE can confer resistance or susceptibility in vivo depending on the drug substrate. Here, we test whether this functional promiscuity of EmrE extends to SMR transporters from three additional human or animal pathogens: SAsmr from Staphylococcus aureus, PAsmr from Pseudomonas aeruginosa, and FTsmr from Francisella tularensis. We find that these SMR homologs can confer either resistance or susceptibility to different toxic substrates in E. coli. This demonstrates that the ability of a single transporter to lead to opposite biological outcomes when transporting different substrates is a general property of the promiscuous multidrug transporters in the SMR family. It also suggests the potential for novel antibiotic development targeting these transporters with small molecules that trigger susceptibility. Such a strategy does not require that the target be the primary mode for antibiotic resistance because the goal is not simple inhibition of activity, but rather activation of an alternative transport function that is detrimental to bacteria.

microbiology↗

bioRxiv2022

Charge neutralization of the active site glutamates does not limit substrate binding and transport by EmrE

EmrE, a small multidrug resistance (SMR) transporter from E. coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of 2 protons:1 cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue while the other remains accessible to bind and transport a proton. Here we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Further, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.

biophysics↗

bioRxiv2021

A new substrate triggers susceptibility by uncoupling a bacterial multidrug resistance efflux pump

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds from the bacterial cytoplasm. Previous biophysical studies of the E. coli SMR transporter EmrE suggested that it should also be capable of performing proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several newly characterized small-molecule substrates in E. coli, including harmane. In vitro experiments show that harmane binding to EmrE triggers uncoupled proton uniport and this protein-mediated dissipation of the transmembrane pH gradient underlies the in vivo phenotype. This leads to synergy with some existing antibiotics, such as kanamycin. Furthermore, this shows that it is possible to not just inhibit multidrug efflux but activate alternative transport modes that are detrimental to bacterial growth and metabolism.

biochemistry↗

bioRxiv2021

Functional phenotyping of small multidrug resistance proteins from Staphylococcus aureus and Francisella tularensis reveals functional homology to EmrE

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells. These transporters were recently divided into two subfamilies: the GdX-like and EmrE-like SMRs. The EmrE-like subfamily of SMRs is predicted to contain transporters that are highly promiscuous in both substrate specificity and mechanism based on extensive characterization of the founding member of this subfamily, EmrE. However, there is only limited functional analysis of other members of this family from pathogenic strains such as Staphylococcus aureus and Francisella tularensis. Here, we use a small compound screen to explore the substrate specificity and diversity of EmrE-subfamily SMRs from these two bacterial species and confirm that they are functionally more like EmrE than the GdX-like subfamily of toxic-metabolite transporters. The results of these experiments lay the foundation for understanding the complex substrate specificity profiles of SMR family transporters and assess the potential for targeting these transporters for future antibiotic development, either broadly or in a species-specific manner.

microbiology↗

bioRxiv2020

Unlocking the reversal potential of solid supported membrane electrophysiology to determine transport stoichiometry

Transport stoichiometry provides insight into the mechanism and function of ion-coupled transporters, but measuring transport stoichiometry is time-consuming and technically difficult. With the increasing evidence that many ion-coupled transporters employ multiple transport stoichiometries under different conditions, improved methods to determine transport stoichiometry are required to accurately characterize transporter activity. Reversal potential was previously shown to be a reliable, general method for determining the transport stoichiometry of ion-coupled transporters (Fitzgerald & Mindell, 2017). Here, we develop a new technique for measuring transport stoichiometry with greatly improved throughput using solid supported membrane electrophysiology (SSME). Using this technique, we are able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx. Our SSME method requires only small amounts of transporter and provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

biochemistry↗

bioRxiv2017

A kinetically-driven free exchange mechanism of EmrE antiport sacrifices coupling efficiency in favor of promiscuity

EmrE is a small multidrug resistance transporter found in E. coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: Not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a new kinetically-driven free exchange model for EmrE antiport that is consistent with these results and recapitulates {Delta}pH-driven concentrative drug uptake. Our results suggest that EmrE sacrifices coupling efficiency for initial transport speed and multidrug specificity.\n\nSIGNIFICANCEEmrE facilitates E. coli multidrug resistance by coupling drug efflux to proton import. This antiport mechanism has been thought to occur via a pure exchange model which achieves coupled antiport by restricting when the single binding pocket can alternate access between opposite sides of the membrane. We test this model using NMR titrations and transport assays and find it cannot account for EmrE antiport activity. We propose a new kinetically-driven free exchange model of antiport with fewer restrictions that better accounts for the highly promiscuous nature of EmrE drug efflux. This model expands our understanding of coupled antiport and has implications for transporter design and drug development.

biophysics↗