Author response:

Public Reviews:

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.

We thank the reviewer for this accurate and thoughtful summary of our work and its broader significance. We agree that the combination of single-molecule FRET with orthogonal validation approaches enables mechanistic resolution of conformational states and transitions that are not accessible by ensemble measurements. In particular, this framework allows direct discrimination of ATP-free and ATP-bound conformations, real-time tracking of transport cycle progression, and identification of transient intermediates in the heterodimeric ABC transporter TmrAB. We further agree that these capabilities support a generalizable strategy for dissecting conformation dynamics in related 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 K_d and its implications on the mechanism.

The apparent K_d,ATP values were determined using two complementary approaches that report on different aspects of the system. Ensemble FRET measurements yielded values of 51° ± 38° µM (TmrAB^NBD), 68° ± 25° µM (TmrAB^PG), and 95° ± 26° µM (TmrAB^PG_EQ), which are in good agreement with previously reported biochemical estimates (~100° µM for TmrAB^EQ) (Stefan et al, 2020). The slightly elevated value observed for the E→Q variant may reflect modest perturbation of nucleotide handling in this slow-turnover background. Notably, the close agreement between labeled and unlabeled variants indicates that fluorophore attachment does not measurably affect ATP binding.

In contrast, smFRET-derived K_d,ATP values (13° ± 1° µM for TmrAB^NBD and 2° ± 1° µM for TmrAB^PG) are systematically lower. This difference likely arises from the difficulty of deconvoluting overlapping FRET populations at sub-K_d,ATP concentrations, particularly for TmrAB^PG, where state assignment is less well separated. Despite this quantitative offset, both approaches consistently indicate ATP saturation well below physiological concentrations and therefore support the same mechanistic conclusion that ATP binding drives conformational switching in TmrAB.

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.

We thank the reviewer for the thoughtful and constructive evaluation of our manuscript and for highlighting the strength of combining structural and single-molecule approaches. We have addressed all major and minor points in detail below and revised the manuscript where appropriate to clarify limitations, justify analysis choices, and improve transparency.

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.

We agree that our conclusions are based on a limited number of FRET reporter pairs, and we now explicitly state this limitation in the revised manuscript. The chosen labeling positions were selected to probe two functionally critical regions—the nucleotide-binding domains and the periplasmic gate—based on prior structural and spectroscopic evidence. While this represents sparse sampling of the full conformational space, it is consistent with typical smFRET studies of membrane transporters, where experimental constraints generally limit the number of simultaneously accessible labeling positions (Asher et al, 2021; Asher et al, 2022; Levring et al, 2023; Wang et al, 2020).

Importantly, both independent reporter variants yield consistent ATP-dependent population shifts, supporting the robustness of the observed trends. We further clarify that additional labeling sites could, in principle, resolve finer structural sub-states; however, given the already limited population separation in the current variants, such extensions would likely provide diminishing returns in state resolvability under the present experimental conditions. This trade-off is now explicitly discussed.

(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?

We thank the reviewer for this careful observation. Low-amplitude subpopulations are occasionally detected in individual histograms; however, they were not included in the quantitative model because they do not meet criteria for reproducibility, amplitude robustness, or structural assignability. Specifically, these features vary between replicates, contribute minimally to total population, and cannot be mapped to structurally or biochemically defined states based on available cryo-EM (Hofmann et al, 2019), DEER/PELDOR (Barth et al, 2018; Barth et al, 2020), or accessible-volume simulations.

Similar minor subpopulations have been reported in smFRET studies and often attributed to photophysical or labeling heterogeneity effects (Asher et al, 2022; Husada et al, 2018). To avoid over-parameterization, we therefore restricted analysis to reproducible, structurally supported states. This rationale is now clarified in the revised manuscript.

(3) Figure 3c (3 mM ATP): Is it truly possible to distinguish the two states in this distribution?

We agree that state separation in the TmrAB^PG variant is limited (ΔE° = °0.11), and we now explicitly acknowledge this constraint in the manuscript. To improve robustness under these conditions, we used a constrained fitting strategy in which the apo-state distribution was fixed from nucleotide-free measurement, reducing parameter degeneracy during fitting of ATP-bound datasets.

While single-molecule trajectory-based approaches such as Hidden Markov Modeling would be ideal for resolving dynamic interconversion, this was not feasible due to the low fraction of dynamic traces at the available temporal resolution. We therefore rely on population-level analysis, which remains consistent across replicates and reporter variants.

Notably, independent measurements from two reporter positions (TmrAB^NBD and TmrAB^PG) yield similar ATP-bound population fractions at saturating ATP concentrations (~77% vs. ~80%), supporting the robustness of the inferred state distribution despite partial overlap.

References

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Hofmann S, Januliene D, Mehdipour AR, Thomas C, Stefan E, Brüchert S, Kuhn BT, Geertsma ER, Hummer G, Tampé R, Moeller A (2019) Conformation space of a heterodimeric ABC exporter under turnover conditions. Nature 571: 580–583. doi:10.1038/s41586-019-1391-0

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Figures and data

Accessible-volume (AV) simulations of LD fluorophores on TmrAB variants.

Quality of TmrAB purification and fluorophore labeling.

FRET capabilities of labeled TmrAB variants.

ATP-induced conformational changes of TmrAB analyzed by smFRET.

Representative smFRET traces of TmrABNBD.

Representative smFRET traces of TmrABPG.

ATP-dependent shifts in smFRET populations of TmrAB.

Identification of the outward-facing open (OFopen) conformation.

Conformational state distribution and catalytic cycle of TmrAB under active turnover.

Representative smFRET traces of TmrAB^NBD.

Representative smFRET traces of TmrAB^PG.

Identification of the outward-facing open (OF^open) conformation.