ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter
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eLife Assessment
The study presents important findings revealing previously unresolved conformational dynamics of the heterodimeric type IV ABC transporter TmrAB using single-molecule FRET. The evidence presented is solid, integrating careful experimental design with computational approaches to uncover states that are typically masked and difficult to detect. The work will be of interest to scientists studying the molecular mechanisms of primary active transport processes.
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Abstract
ATP-binding cassette (ABC) transporters are essential molecular machines whose conformational dynamics have largely been inferred from ensemble-averaged measurements. Resolving dynamic heterogeneity and transient intermediates, however, requires single-molecule approaches. Here, we use single-molecule Förster resonance energy transfer (smFRET) to directly monitor conformational changes of the heterodimeric type IV ABC transporter TmrAB, a functional homolog of the human antigen transporter TAP. Fluorophores positioned at the nucleotide-binding domains and the periplasmic gate were validated by accessible-volume simulations, fluorescence lifetimes, and ensemble FRET, demonstrating that these reporters reliably track conformational transitions. Single-molecule analysis distinguishes ATP-free and ATP-bound states and reveals ATP-dependent population shifts from nucleotide-free to physiological ATP concentrations. Probing conformational dwell-times further uncovers an unexpectedly long ATP-bound dwell time of ∼300 ms. Using complementary stabilization strategies–including a slow-turnover variant, Mg²⁺ depletion, or substrate trans-inhibition–we resolve a previously hidden outward-facing open state that rapidly interconverts with occluded intermediates under turnover conditions. These results provide the first single-molecule characterization of TmrAB and establish a general framework for dissecting ATP-coupled conformational dynamics in heterodimeric ABC transporters.
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eLife Assessment
The study presents important findings revealing previously unresolved conformational dynamics of the heterodimeric type IV ABC transporter TmrAB using single-molecule FRET. The evidence presented is solid, integrating careful experimental design with computational approaches to uncover states that are typically masked and difficult to detect. The work will be of interest to scientists studying the molecular mechanisms of primary active transport processes.
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Reviewer #1 (Public review):
Summary:
Pecak et al have deciphered the conformational dynamics of a heterodimeric model ABC transporter, TmrAB, a functional homolog of the human antigen transporter TAP, using single-molecule Forster resonance energy and fluorophores attached to residues at either nucleotide binding domains or periplasmic gate. The analysis not only differentiated ATP-free and bound states but also enabled the real-time monitoring of protein conformational changes, precisely dissecting transport cycles and resolving transient intermediates. This study is absolutely significant in providing and establishing a general pipeline delineating the conformational dynamics in heterodimeric ABC transporters.
Strengths:
The scientific study is very well documented for experimental design, results, and conclusions supported by the …
Reviewer #1 (Public review):
Summary:
Pecak et al have deciphered the conformational dynamics of a heterodimeric model ABC transporter, TmrAB, a functional homolog of the human antigen transporter TAP, using single-molecule Forster resonance energy and fluorophores attached to residues at either nucleotide binding domains or periplasmic gate. The analysis not only differentiated ATP-free and bound states but also enabled the real-time monitoring of protein conformational changes, precisely dissecting transport cycles and resolving transient intermediates. This study is absolutely significant in providing and establishing a general pipeline delineating the conformational dynamics in heterodimeric ABC transporters.
Strengths:
The scientific study is very well documented for experimental design, results, and conclusions supported by the experimental data. The authors have determined the conformational dynamics of TmrAB across different ATP concentrations, including physiological ones, and resolved an outward open state and other conformational states consistent with previous cryoEM and DEER studies.
Weaknesses:
The scientific study needs a bit of in-depth analysis with respect to consistency in Kd and its implications on the mechanism.
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Reviewer #2 (Public review):
In their manuscript entitled 'ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter', Pečak et al. use elegant single-molecule FRET experiments in detergent to investigate the heterodimeric ABC transporter TmrAB. By combining simulations of the transporter's accessible volume with elegant trapping strategies, the authors identify an unresolved outward-facing open state and conclude that it is usually obscured by a rapidly interconverting ATP-bound ensemble. Overall, the study demonstrates that smFRET can resolve the short-lived intermediate states of TmrAB and potentially other ABC transporters that are obscured in ensemble measurements.
It is a very interesting study that highlights the power of combining high-resolution structural information with spectroscopic …
Reviewer #2 (Public review):
In their manuscript entitled 'ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter', Pečak et al. use elegant single-molecule FRET experiments in detergent to investigate the heterodimeric ABC transporter TmrAB. By combining simulations of the transporter's accessible volume with elegant trapping strategies, the authors identify an unresolved outward-facing open state and conclude that it is usually obscured by a rapidly interconverting ATP-bound ensemble. Overall, the study demonstrates that smFRET can resolve the short-lived intermediate states of TmrAB and potentially other ABC transporters that are obscured in ensemble measurements.
It is a very interesting study that highlights the power of combining high-resolution structural information with spectroscopic approaches. I have three major points and a few minor criticisms.
Major points:
(1) The main weakness is that the authors base their conclusions on a very limited set of FRET pairs. While TmrAB has been extensively studied in terms of its structure, the authors should at least acknowledge this limitation more clearly.
(2) Most smFRET distributions were fitted with one, two, or three Gaussians. However, in several cases, additional populations with noticeable amplitudes appear to be present (e.g., Figure 3c at 0.1 mM and 3 mM ATP; Figure 4a, apo; Figure 4c, 0.3 mM R9L). Could the authors clarify why these populations were not included in the analysis?
(3) Figure 3c (3 mM ATP): Is it truly possible to distinguish the two states in this distribution?
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