Projects in the Henzler-Wildman Lab investigate how the structure and dynamics of integral membrane proteins contribute to their function. We are particularly interested in the molecular mechanism of transporters and channels. Since large scale conformational changes between different states is a key part of the proposed mechanism for transporters and channels, these are ideal systems for studying the role of protein motion in proper function of integral membrane proteins. We have also begun to work on several SARS-CoV-2 proteins in collaboration with colleagues. We use NMR and other biophysical methods to characterize the timescale, amplitude and direction of structural changes, and combine this data with functional assays to study the mechanism of secondary active transport, multidrug recognition, ion selectivity, channel gating, temperature sensing and allosteric regulation of membrane protein function.
EmrE and Other Small Multidrug Resistance (SMR) Transporters
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Measuring functional dynamics of integral membrane proteins
We used solution NMR dynamics experiments to quantitatively measure the exchange between inward- and outward- facing states of the Small Multidrug Resistance (SMR) transporter EmrE in bicelles. This was possible using our optimized protocol for bicelle solubilization of integral membrane proteins and the unique ability of NMR to simultaneously measure structural (chemical shift), kinetic (rates), and thermodynamic (populations) parameters at multiple sites across the protein. Combining the NMR data with single molecule FRET experiments under matching conditions resolved a long-standing controversy surrounding the functional topology of the EmrE homodimer. Our data demonstrated that antiparallel EmrE switches between states open to opposite sides of the membrane, establishing the antiparallel dimer as the functionally relevant form. Blocking this conformational exchange without perturbing the structure blocks transport activity in its native E. coli, providing the most direct experimental validation of the alternating access mechanism for transport. This work helped established the use of solution NMR methods to measure integral membrane protein dynamics. This represents an important advance, since motions are critical for many important biological functions in transport and signaling, but measurement of integral membrane protein dynamics has been challenging using traditional structural biology methods. More recently, we have discovered a point mutant of EmrE that significantly reduces alternating access rates without disrupting substrate binding. This novel mutant provides new insight the molecular features controlling how and when functionally important conformational transition occur in membrane proteins.
Morrison, E. A.*, Dekoster, G. T.*, Dutta, S., Clarkson, M., Vafabkhsh, R., Bahl, A., Kern, D., Ha, T., and Henzler-Wildman, K. A. (2012) “Antiparallel EmrE Exports Drugs by Exchanging Between Asymmetric Structures” Nature. 481, 45-50.
Dutta S., Morrison, E. A. and Henzler-Wildman, K. A. (2014) “Blocking Dynamics of the SMR Transporter EmrE Impairs Efflux Activity” Biophysical Journal. 107, 613-620.
Morrison, E. A., Henzler-Wildman, K. A. (2012) “Reconstitution of Integral Membrane Proteins into Isotropic Bicelles with Improved Sample Stability and Expanded Lipid Composition Profile.” Biochimica et Biophysica Acta Biomembranes. 1818, 814-820.
Wu, C., Wynne, S., Thomas, N., Uhlemann, E., Tate, C. and Henzler-Wildman, K. (2019) “Identification of an Alternating-Access Mutant of EmrE with Impaired Transport” Journal of Molecular Biology. 431, 2777-2789.
Transport mechanism of EmrE and other small multidrug resistance transporters
Protein conformational change is required for active transport, allowing alternating access to either side of the membrane in order to move a substrate “uphill”. The energy source for secondary transporters is the “downhill” flow of another substrate, often protons. Our research investigates the protein dynamics central to the transport process and the coupling between substrates that drives active transport.
NMR is uniquely able to monitor proton binding through the impact that protonation has on the chemical shift of nearby nuclei. This allows direct detection of protonation events independently of any coupled effect on substrate binding or transport. Using pH titrations with and without substrates we made several important discoveries. First, the E14 residues in the EmrE homodimer have different pKa values. In retrospect, this is not surprising because of the asymmetric structure of the antiparallel homodimer places each E14 in a unique environment. Second, our NMR data that unambiguously shows that EmrE can bind both proton and drug substrates simultaneously and it can switch from open-in to open-out with both bound. This is the behavior expected of a symporter not an antiporter. Kinetic simulations show that proton-coupled transport can be achieved without invoking the usual constraints on substrate binding and alternating access. Most convincingly, EmrE can perform both H+/drug+ antiport other types of transport in proteoliposome transport assays. These results expand the promiscuity of EmrE transport from multiple substrates to include multiple proton-coupling stoichiometries for a single substrate. Third, we discovered that protonation of the C-terminal histidine residue is coupled to drug- and proton-binding in the primary binding site at E14, raising questions about the potential mechanistic importance of the C-terminal tail that is not well resolved in any of the current EmrE structures.
We have developed a novel solid-supported-membrane electrophysiology assay to rapidly and reliably measure transport stoichiometry under a variety of conditions that will enable more thorough characterization of transport mechanisms. Ongoing work uses this assay and NMR methods to study how different mutants and substrates affect the structure, dynamics, and function of EmrE to refine the molecular mechanism underlying coupled transport and drug resistance.
Robinson, A. E., Thomas, N. E., Morrison, E. A., Balthazor, B. M., and Henzler-Wildman, K. A. (2017) “New Free-Exchange Model of EmrE Transport” Proceedings of the National Academy of Sciences USA 114, E10083-E10091. PMCID: PMC5703289
Morrison, E. A., Robinson, A., Liu, Y. and Henzler-Wildman, K. A. (2015) “Asymmetric protonation of EmrE” Journal of General Physiology. 146:445-461. PMCID: PMC4664823.
The C terminus of the bacterial multidrug transporter EmrE couples drug binding to proton release.” Journal of Biological Chemistry 293:19137-19147. PMCID: PMC6295725
Thomas, N. E., Feng, W., and Henzler-Wildman, K. A. (2021) “A solid-supported membrane electrophysiology assay for efficient characterization of ion-coupled transport” Biol. Chem. 297:101220. PMCID: PMC8517846.
How does EmrE recognize and transport such a diverse set of substrates?
We have shown that properties of the small molecule substrate determine its rate of transport by altering the open-in/open-out exchange rate of the transporter. Do substrate properties control not only the rate, but also the type of transport? We are using the naturally diverse set of substrates for this promiscuous multidrug transporter to study what chemical features make a “good” substrate that is efficiently transported by EmrE and the correlation between specific chemical properties and the rate of transport. We have performed kinetic simulations to demonstrate how different substrates could trigger a shift in the behavior of a promiscuous transporter from proton/drug antiport to other modes of transport, such as proton/drug symport.
Most recently, we have obtained structural restraints to locate a substrate within the primary EmrE binding site, providing the first experimental distance restraints between an EmrE substrate and specific amino acid side chains important for substrate binding and recognition. Changes in these substrate-protein distance restraints at high and low pH suggest why drug binding is pH-dependent and how simultaneous proton binding weakens drug affinity. We are continuing to use structure, mutagenesis and functional assays to probe how different substrates interact with EmrE to trigger different rates of alternating access and different modes of transport.
Morrison, E. A. and Henzler-Wildman, K. A. (2014) “Transported substrate determines exchange rate in the multidrug resistance transporter EmrE” Journal of Biological Chemistry. 289, 6825-6836. PMCID: PMC3945343.
Hussey, G. A., Thomas, N. E., and Henzler-Wildman, K. A. (2020) “Highly coupled transport can be achieved in free-exchange transport models.” Journal of General Physiology. 152:e201912437. PMCID: PMC7034097.
Shcherbakov, A. A., Hisao, G., Mandala, V. S., Thomas, N. E., Soltani M., Salter E. A., Davis Jr., J. H., Henzler-Wildman, K. A., and Hong, M. (2021) “Drug-binding structure and drug dynamics of the bacterial transporter EmrE in lipid bilayers” Nature Communications. 12:172. PMCID: PMC7794478.
High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport” Nature Communications. PMCID: PMC8857205.
What is the native function of SMR transporters? How can we target this family?
We are performing resistance/susceptibility assays to better characterize the substrate profile of SMR transporters and determine whether any SMR homologs naturally confer susceptibility rather than resistance to any compounds. We are investigating what compounds are transported by SMR transporters in vivo under different stress conditions where expression of EmrE or other SMR transporters impacts bacterial growth and survival.
Are EmrE or other SMR transporters viable targets for antibiotic development?
The SMR transporters other than EmrE have not been widely studied. Recent research suggests that SugE and many of the “SMR” family may not even function as drug resistance transporters, but are instead guanidinium exporters. Our recent work suggests that EmrE can act as both a proton-coupled symporter and antiporter of different substrates. This would result in susceptibility (symport) as well as resistance (antiport) since the proton motive force is always inwardly directed in bacteria. This is unprecedented for a transporter and is functionally significant because of the potential to control transport direction by manipulating properties of the small molecule, opening up the possibility of designing drugs that are selectively imported into bacteria by the SMR transporters.
NaK, a bacterial non-selective cation channel
NaK provides an ideal model system for investigating ion selectivity and for comparison with the potassium-selective KcsA channel. Although NaK is a non-selective cation channel, two point-mutations convert it into a potassium-selective channel. We are using NMR to study how atomic-level motions influence ion selectivity and the coupling between channel gates that underlies inactivation in the bacterial NaK channel. Functional studies will test whether the insights obtained from our biophysical studies have the proposed effects on the activity of this bacterial channel.
Brettmann, J. B., Urusova, D., Tonelli, M., Silva, J. R. and Henzler-Wildman, K. A. (2015) “Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel” Proceedings of the National Academy of Sciences USA. 112:15366-15371. PMCID: PMC468759.
Lewis, A., Kurauskas, V., Tonelli, M. and Henzler-Wildman, K. A. (2021) “Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel” Commun. 12:6225. PMCID: PMC8553846.
SARS-CoV-2 protein structure and dynamics
We are studying the structure and dynamic of several non-structural proteins that are found in several different complexes where they have different conformations. Our goal is to understand the structure of these proteins alone and in complex with different partners, and how they transition between these different conformational states. We are also working on the structure of SARS-CoV-2 proteins in the membrane for which there is less structural data currently available.
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.” Frontiers in Molecular Biosciences. 8:653148. PMCID: PMC8141814.
1H, 13C, and 15N backbone and side chain chemical shift assignments of the SARS-CoV-2 non-structural protein 7.” Biomolecular NMR Assignments 15:73-77. PMCID: PMC7678775.