Tracking multiple conformations occurring on angstrom-and-millisecond scales in single amino-acid-transporter molecules

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The AdiC transporter facilitates the movement of arginine and its metabolite across the membrane of pathogenic enterobacteria, enabling them to evade a host’s highly acidic gastric defense barrier to reach the intestines. Like other transporters, AdiC undergoes a series of necessary conformational changes. Detection of these changes, which occur on angstrom-and- millisecond scales, remains extremely challenging. Here, using a high-resolution polarization-microscopic method, we have successfully resolved AdiC’s four conformations by monitoring the emission-polarization changes of a fluorophore attached to an α-helix that adopts conformation-specific orientations and, furthermore, quantified their probabilities in a series of arginine concentrations. The K D values determined for arginine in four individual conformations are statistically comparable to the previously reported overall K D determined using isothermal titration calorimetry. This demonstrated strong resolving power of the present polarization-microscopy method will enable an acquisition of the quantitative information required for understanding the expected complex conformational mechanism underlying the transporter’s function, as well as those of other membrane proteins.

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  1. eLife assessment

    This paper presents a single-molecule polarization microscopy study aimed at monitoring the arginine/agmatine antiporter AdiC as it transiently exchanges between conformational states. This approach measures how a bis-TMR fluorophore anchored onto helix 6a changes its orientation in the microscope, and the authors identify four states that they propose correspond to the key steps in the transport cycle (inward-open, inward occluded, outward occluded and outward open). This is a cutting-edge and challenging approach that sets the stage for direct measurements of conformational equilibria and will thus be of interest to anyone studying transport mechanisms. However, additional investigation is required to validate the robustness of the post-processing of the single-molecule data to yield the four-state model compared to alternate models, to test the robustness of the data with transport mutants/conditions that would slow or eliminate states, and to consolidate transitions that are observed that conflict with previous observations of obligatory coupling in AdiC.

  2. Reviewer #1 (Public Review):

    For membrane transporters, the factors that define transport cycle state equilibria and kinetics remains a major question. In contrast to ion channels where electrophysiological single-channel recordings reveal transitions between states, this has not been possible for slower transport proteins and so this information must be extracted from bulk transport behavior. However, recent single-molecule microscopy studies, such as FRET, have provided a new way of identifying transitions between conformational ensembles and connecting this to transport behaviors. However, the resolution of FRET can be limiting in that it requires multiple labeling with large fluorophores that have their own freedom to move, thus reducing the ability to detect small conformational changes. In the present study, Zhou et al. address this by using a different single-molecule approach of polarization microscopy, and investigate the small conformational changes associated with the AdiC arginine/agmatine antiporter from the APC super-family of transport proteins. Here, they anchor bis-TMR-maleimide onto helix 6, a part of the protein that has been identified to change orientation in the different crystal structures of AdiC and other APC homologues in inward, outward and occluded states. By "fixing" the protein onto microscopy slides, they are able to detect the change in polarization angles of the emitted fluorescence and map that onto relative changes in helix 6 orientation. Analyzing these data, they propose a model of four states that exchange in equilibrium, with and without the substrate, setting the stage for quantifying equilibrium constants and kinetics for a detailed mapping of the transport cycle, presented in an accompanying article.

    This is certainly a cutting-edge approach that offers the potential to resolve the equilibrium reactions between small conformational changes and thus has the potential to push forward the mechanistic and quantitative investigation of membrane transport. However, at this point the studies require further validation on several levels. This includes an independent investigation of whether the protein being studied (i.e. with all tags, mutations, labeling, nanodisc solubilzation) confers the same substrate binding and transport behavior that has been reported previously, and is being used as comparison data here. In addition, there is some concern that the anchoring of the protein may bias conformational equilibria in some way and so it would be worthwhile to map out if this effect is limiting by changing linker lengths, within a range where it is still possible to resolve changes in polarization angles. Finally, the results are very dependent on the post-processing of the single-molecule trajectories that include changepoint analysis, averaging and clustering algorithms, yet there is little data provided to examine the robustness of each of these steps in the ultimate determination of the four-state model. While the observation that some of the states identified show a linkage to the arginine substrate, further validation along the lines mentioned above are required before a full analysis of the transport cycle is rationalized.

  3. Reviewer #2 (Public Review):

    The antiporter AdiC is a member of the amino-acid and polyamine organocation (APC) transporter superfamily. It imports the single-charged arginine (Arg+) and exports the double-charged agmatine (Agm2+). Thus, it increases the intracellular pH, helping some pathogenic enterobacteria survive in acidic environments. The APC transporters are known to sample 4 major conformations in the transport cycle. Monitoring the conformational transitions is important for understanding the transport mechanism, but methods detecting multi-state conformational changes are very limited. The authors use high-resolution polarization microscopy to resolve 4 different states in substrate-free (Apo) or substrate-bound conditions. This work further demonstrates the power of fluorescence polarization microscopy in studying protein dynamics. The authors introduced an interesting normalization step in data processing to average results obtained for different protein particles. However, the 4 states could be identified from single traces and the normalization from trace to trace could not be done without the pre-identified states on single traces. Thus, the improvement provided by the normalization compared to the published work (NSMB 2019a, 2019b, 2019c) is relatively limited.

  4. Reviewer #3 (Public Review):

    In this work, Zhou et al. employed the polarization microscope (PM) method to track the orientations of helix 6a in the bacterial amino-acid transporter AdiC. It is very impressive that the authors were able to optimize the technique to achieve an overall resolution of 5{degree sign} for detecting changes in the inclination and rotation angles (𝜃 and 𝜓). However, I am deeply concerned about how the authors linked PM-detected conformational states to the structural states obtained using crystallography. Overall, I think it was an overstatement that the work resolved the equilibrium conditions for the major states in AdiC's transport cycle, and I urge the others to be more transparent with the readers about the limitations of their technique and be more thorough in considering alternative interpretations.

  5. Reviewer #4 (Public Review):

    In this paper, Zhou et al. propose a polarization microscope for measuring the emission polarization of bifunctional rhodamine molecules attached to AdiC transporters. The polarization is used to resolve the orientation of the fluorophores, which allows the authors to successfully resolve the four conformations of AdiC at a temporal resolution of tens of milliseconds. The measured orientation for each conformation is validated with the results using crystallography.

    Overall, I believe the paper is well written and demonstrates a great application for orientation imaging using polarized microscopes. Detailed experimental procedures, calibrations, and mathematical frameworks are included. I have the following recommendations to improve the manuscript.

    1. On page 20, the authors note that they set a threshold to filter out molecules whose total intensity varies during the measurements. The statement that "while fluorescence intensity is expected to vary among different polarization directions, the total intensity should be essentially invariant" is not true. Since the authors use TIRF illumination to excite the molecules, the excitation polarization component along the tilting direction (e.g., along the y-axis) of the excitation is 0, i.e., molecules oriented along that direction (e.g., y-oriented) will be excited less effectively compared to other orientations.

    2. Could the authors provide more details regarding how the clusters are ranked? The authors note that C1-C4 are "ranked according to the values of both angles". It is not clear to me how this is done. Also, what is the range of the measured theta_L and phi_L? And how is the warping of the spherical coordinates handled in the ranking process, e.g., a change from 350 deg to 10 deg is +20 deg or -340 deg.

    3. Is the k-means clustering also based on the distance in the Cartesian space, similar to the state identification?