The active-site glutamate 14 residues of the homodimeric multidrug transporter EmrE have distinct macroscopic pKa values, raising new questions regarding the coupling of proton import to drug efflux.

Secondary active transporters couple the transport of an ion species down its concentration gradient to the uphill transport of another substrate. Despite the importance of secondary active transport to multidrug resistance, metabolite transport, and nutrient acquisition, among other biological processes, the microscopic steps of the coupling mechanism are not well understood. Often, transport models illustrate coupling mechanisms through a limited number of “major” conformations or states, yet recent studies have indicated that at least some transporters violate these models. The small multidrug resistance transporter EmrE has been shown to couple proton influx to multidrug efflux via a mechanism that incorporates both “major” and “minor” conformational states and transitions. The resulting free exchange transport model includes multiple leak pathways and theoretically allows for both exchange and cotransport of ion and substrate. To better understand how coupled transport can be achieved in such a model, we numerically simulate a free-exchange model of transport to determine the step-by-step requirements for coupled transport. We find that only moderate biasing of rate constants for key transitions produce highly efficient net transport approaching a perfectly coupled, stoichiometric model. We show how a free-exchange model can enable complex phenotypes, including switching transport direction with changing environmental conditions or substrates. This research has broad implications for synthetic biology, as it demonstrates the utility of free-exchange transport models and the fine tuning required for perfectly coupled transport.

NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps associated with its practice have made the approach daunting for non-specialists. We introduce an NMR platform that makes biomolecular NMR spectroscopy much more accessible by integrating tools, databases, web services, and video tutorials that can be launched by simple installation of NMRFAM software packages or using a cross-platform virtual machine that can be run on any standard laptop or desktop computer. The software package can be downloaded freely from the NMRFAM software download page (http://pine.nmrfam.wisc.edu/download_packages.html), and detailed instructions are available from the Integrative NMR Video Tutorial page (http://pine.nmrfam.wisc.edu/integrative.html).

Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alternating access. We performed extensive scanning mutagenesis by changing every amino acid residue to Val, Ala, or Gly, and then screening the drug resistance phenotypes of the resulting mutants. We identified EmrE mutants that had impaired transport activity but retained the ability to bind substrate and further tested their alternating access rates using NMR. Ultimately, we were able to identify a single mutation, S64V, which significantly reduced the rate of alternating access but did not impair substrate binding. Six other transport-impaired mutants did not have reduced alternating access rates, highlighting the importance of other aspects of the transport cycle to achieve drug resistance activity in vivo. To better understand the transport cycle of EmrE, efforts are now underway to determine a high-resolution structure using the S64V mutant identified here.

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