The Transport Cycle in Neurotransmitter Uptake Systems update, year 1

Summary of Progress

A. Specific Aims

Aim 1: To identify and characterize computationally and experimentally mutations and substrate/ligand settings that alter the conformational equilibrium of LeuT (between the states allowing access from the extracellular and intracellular sides, respectively). The combination of approaches will be applied iteratively to simulate and measure transport, binding, and dynamics (e.g., with EPR measurements of accessibility of engineered cysteines). This will establish experimental conditions that enrich differentially conformational states that we have already identified in the transport cycle.

Aim 2: To characterize the transitions between states and identify large scale rearrangements associated with the distinct conformational states established in Aim 1, using both continuous wave (CW) and double electron-electron resonance (DEER) EPR spectroscopy to measure spin-spin distances.

B. Studies and Results

Bridge 3, which now completed its first year of activity, comprises three synergistic components combining experimental and computational approaches in a collaboration between the Javitch, Mchaourab, and Weinstein labs. The Javitch lab has focused on functional characterization of LeuT, which, in conjunction with computational studies in the Weinstein lab, has uncovered a novel mechanism that depends upon the functional coupling of two substrate sites, the S1 and S2 sites, the latter of which is masked in the available crystal structures. Supported by another mechanism, the lab is also pursuing single molecule FRET (smFRET) experiments to probe the dynamics of LeuT in parallel to the computational simulations. This research is enhancing the scope of investigation and leverages the Glue grant context to disseminate smFRET data and mechanistic interpretations among the Bridge 3 collaborators and the Cores (see below). One example is the unprecedented opportunity that this Bridge provides to compare EPR-based findings with those from smFRET studies (see Abstracts below). The Mchaourab laboratory has focused within this first year of funding on the dynamic information obtained from a series of pairs of spin labels (48 pairs) strategically placed on both the extracellular and intracellular sides of LeuT to capture the expected opening and closing movements of these regions that must occur during the transport cycle. Together with the computational analysis methods, the approaches can be calibrated and interpreted with greater accuracy so as to advance our insights into the dynamics of this model system for secondary transport with an unprecedented combination of tools. The Javitch lab has also identified substrates, inhibitors, and mutations that bias the transporter conformation. Parallel computational studies in the Weinstein lab using various forms of biased and unbiased MD simulations have led to mechanistic insights that can be further evaluated using collaborative experiments with both smFRET and EPR. For example, the EPR studies in the Mchaourab lab have enabled us to identify and quantify conformational changes that are associated with various functional states of the transporter in a manner consonant with the computational simulations. The functional characterization and the computational simulations are applied together to evaluate the results from the EPR studies in an iterative manner that takes advantage of our findings regarding the ability of the Na+ ions and the transported substrates Leu and Ala, and of specific mutations, to bias the conformation of the transporter toward various functional states. To this end, the Mchaourab lab has made great progress this year in establishing robust methods for studying LeuT by DEER both in detergent and reconstituted into a lipid environment in nanodiscs (see Highlights below).

A major issue that we addressed this year was the controversy regarding the substrate binding and stoichiometry of LeuT. We discovered that binding to the S2 site can be elusive because LeuT is very sensitive to elevated detergent concentrations so that common protein purification methods can obscure the S2 site and thus impair the functional mechanism that drives transport (see Highlights below). This work has been accepted for publication in Nature Structural and Molecular Biology.

C. Collaborations

The Bridge has been enhanced by collaborations with the Computational Modeling Core through the work of the Weinstein lab, by Core 3 – the Synthetic Antigen Binder Core, in the form of collaboration between the Javitch and Kossiakoff labs, and by Core 1 – the Protein Expression Core, in the form of collaboration between the Mchaourab and Dotsch laboratory to achieve cell-free expression of LeuT. We have contributed new methods for the quantitative study of membrane protein interactions, and have screened for Fabs that bind LeuT in particular conformations and identified a panel of binders that will be characterized during the coming year. The plan is to use them to stabilize transporter conformations for the EPR studies in Bridge 3, as well as for crystallography and smFRET studies in the Javitch lab. Cell-free expression of LeuT directly into micelles or nanodiscs will circumvent many of the pitfalls and potential purification artifacts and simplify the production of mutants for EPR analysis.

D. Plans

In the coming year we plan to identify conformational changes at the intracellular and extracellular sides of LeuT that are associated with transport by completing an extensive series of DEER studies. These studies will continue to be guided by SMD and MD studies that are continuing in the Weinstein lab, as well as structural hypotheses that results from analyses of the resulting trajectories. In the coming year, the parallel computational studies aim to achieve a mechanistic description of the conformational transitions in LeuT in different environments (e.g. lipid bilayers and micelles of different lipid/detergent compositions) and to elucidate structural and functional determinants that determine the role of the membrane in the function and dynamics of LeuT. Special attention will be given to the generalizable insights about specific contributions from various membrane components and the effect of membrane remodeling on the functional mechanisms. The design of specific LeuT/detergent/lipid ratios in the planned simulations, and the probing of the mechanistic hypotheses pursued in the computational modeling and simulations, are enabled by the ongoing experimental studies in the Javitch and Mchaourab labs. The methodology for quantifying the membrane deformation by protein-membrane interactions, and the effects on membrane protein function are calculated with methods developed in the Weinstein lab for dissemination through the Computational Modeling Core, as they represent general approaches for the computational study of membrane protein function in the context of the cell membrane.

Thus, the activity of the consortium has already created an active context for exploration of new methods as well as the application of the technology and insights we have developed within this Bridge to the study of various membrane proteins. An additional example is the collaboration between the Mchaourab lab and Dr. Dötsch that has been developing approaches for in vitro translation of LeuT to expedite our ability to screen many mutants without laborious expression and purification (see Highlights below). The Mchaourab lab in collaboration with Dr, Glaubitz is also attempting to use solid state NMR to examine directly the binding of Leu to the S2 site (See Highlights below). The Mchaourab lab has also carried out preliminary studies with ApcT, a transporter from the amino acid, polyamine, and organocation (APC) transporter family that has a LeuT-like structural fold, in an attempt to expand our findings to other transporter families.

Research Highlights

Click to find out more about each research highlight (RH).

RH #1. Experimental conditions can obscure the simultaneous binding of the two substrate molecules required for LeuT-mediated Na+-coupled transport

RH #2. Solid State NMR studies of leucine binding to LeuT

RH #3. Preliminary computational simulations of LeuT in detergent

RH #4. Monitoring conformational changes in the extracellular region of LeuT using DEER

RH #5. Monitoring conformational changes in the intracellular region of LeuT using DEER

RH #6. Cell-free expression of LeuT

RH #7. Conformational cycle of the organo-cation transporter ApcT