1. Evaluation Summary:

    The paper describes novel structures of a protein recently reported to function as a mechanosensitive ion channel. Surprisingly, the structures and functional data rather support the formerly suggested role of this protein in lipid metabolism. The paper is of relevance for ion channel field and for those interested in fatty acid metabolism.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Yao Rang and collaborators find that heterologous expression of TMEM120A from mouse and human in cells that lack Piezo1 does not result in poke- or stretch-activated currents in whole cells or excised patches, and further detect no mechano-sensitive currents when the purified human protein is reconstituted in giant unilamellar vesicles. Together with high-quality positive controls with Piezo1, Piezo2 and TMEM63a, the results presented here call into question a previous proposal (Beaulieau-Laroche et al., Cell 2020) that TMEM120A functions as the long sought-after mechano-activated channel responsible for detecting painful touch.

    Although the evidence supporting a channel function for TMEM120A is not strong, it remains to be ruled out that the discrepancies between the two studies arise from the different methods that were used to deliver the mechanical stimuli, as mentioned by the authors in the Discussion, or from the C-terminal mCherry tag attached to human TMEM120A in this study that was not present in the construct used by Beaulieau-Laroche et al.

    Upon determination of the structure of full-length human TMEM120A in nanodiscs using cryo-EM, the authors find that the protein forms a dimer with six transmembrane helices per subunit and a cytosolic N-terminal coiled coil domain. Surprisingly, the authors find a density attributable to coenzyme-A (CoASH) located within a highly conserved cytosolic cavity at the transmembrane domain. The authors provide evidence from mass-spectrometry and isothermal titration calorimetry (ITC) to demonstrate that CoASH binds TMEM120A, and solidify their conclusions by showing that mutation of a residue close to the CoASH density in TMEM120A disrupts binding measured by ITC. The authors show that a potential ion-conduction pathway in their TMEM120A structure would be occluded by CoASH on the cytosolic side and on the extracellular side by a series of not well-conserved residues. Finally, the authors solve a structure in detergents where no density for CoASH is observed, as expected from spectroscopic data showing that detergent-reconstitution results in loss of CoASH binding. In this structure, a conformational change is suggested to occur on the cytosolic cavity entrance where a loop becomes reoriented to occlude the cavity that is otherwise occupied by CoASH. Together, the data presented paints an intriguing alternative for the function of TMEM120A proteins with a role in metabolism or CoA transport.

    Although the main conclusions are well supported by the evidence, it is challenging to appraise many of the interesting structural observations pointed out by the authors because the experimental data (i.e. the density) is in most cases not depicted. Some of these observations for which only the model rather than the experimental data is shown include the hinge-like motif at the dimer interface (Fig. 3C), the CoASH binding site (Fig. 4E and Fig. 4 Supplement 1C), the difference in the conformation of the IL5 loop between the apo and CoASH-bound structures (Fig. 5), the extracellular constriction of the possible ion-conduction pathway (Fig. 4 Supplement 1D and Fig. 5 Supplement 2), as well as the comment that the observed density in the structures cannot accommodate other CoA-derivatives, for which data is not shown. The relatively low resolution at which the data were obtained raises concerns regarding many of these detailed observations.

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  3. Reviewer #2 (Public Review):

    In this manuscript, Rong et al., address the possibility that TMEM120A, also known as TACAN, might not be a mechanosensitive ion channel. They use extensive electrophysiological characterization to convincingly show that cells heterologously transfected with either human or mouse TMEM120A do not exhibit mechanosensitive currents above background in cell lines or when purified and reconstituted in giant unilamellar vesicles. They also solve the cryo-EM structure of TMEM120A and find that it does not resemble an ion channel (i.e., there is no obvious pore). Interestingly, there is a density consistent with coenzymeA in the structure, thus suggesting an alternative function for TMEM120A. Further evidence for this interaction is shown through biochemical analysis, as disrupting a residue proposed to form a π−π stacking interaction between TMEM120A and coenzymeA reduces the binding affinity as assayed by ITC.

    Overall, the impact of this manuscript is extremely high, as it refutes a recent report that TMEM120A/TACAN is a high-threshold (pain) mechanosensitive ion channel, and further suggests an alternative function for this protein. The experiments are extremely carefully done, and the authors attempted to replicate the electrophysiological function of TMEM120A with high numbers and with appropriate positive and negative controls. The inclusion of structures (Coenzyme-A bound and apo) and corresponding biochemical analyses provide strong support for the direct binding of Coenzyme-A by TMEM120A.

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  4. Reviewer #3 (Public Review):

    TMEM120A protein was recently reported to mediate mechanosensitive currents in response to painful stimuli. In the present manuscript, the authors aimed to elucidate TMEM120A mechanism of action by solving the structures and complementing them with functional characterization. In contrast to the recent report, the authors could not observe TMEM120A-mediated currents in response to mechanical stimuli neither in transfected cells nor in liposomes. Furthermore, the structure of human homolog HsTMEM120A revealed a co-purified endogenous ligand, which was shown to be coenzyme A (CoASH). The authors went on to solve the structure in the absence of CoASH, revealing a different conformation of HsTMEM120A. Together, structural and functional data point towards a conclusion that TMEM120A might not be a mechanosensitive channel, but might rather be important for fatty acid metabolism. The conclusions of the manuscript are supported by the presented data.

    Strengths:

    1. The authors conclusively show that TMEM120A does not mediate poking- or stretch-induced currents when compared to well-characterized mechanosensitive channels Piezo1 and TMEM63a.

    2. The authors employed several approaches to confirm the identity of the co-purified ligand. Firstly, the presence of CoASH in the purified protein sample was confirmed by mass spectrometry. Secondly, the binding of CoASH to TMEM12A was confirmed by ITC. Thirdly, using the obtained structure the authors identified CoASH-interacting residues and show that mutating one of the key residues (Trp193) reduced TMEM120A affinity for its ligand.

    3. The observation that CoASH dissociates from TMEM120A during size exclusion in detergent, but not in lipid environment allowed to solve a ligand-free TMEM120A structure, which revealed a different conformation at the entrance to CoASH-binding site and is possibly relevant for the mechanism of action.

    Noteworthy, 3 other studies (Niu et al., 2021, Xue et al., 2021, Ke et al., 2021) independently arrived at the conclusion that TMEM120A is probably not a mechanosensitive channel, further supporting the results of the present study.

    Weaknesses:

    Despite the advances presented in this manuscript, physiological function of TMEM120A and its mechanism of action remain obscure, other than that it is probably not a mechanosensitive channel. However, the goal of the authors was to understand how TMEM120A might mediate mechanosensitive currents, while establishing its role in lipid metabolism is outside of the scope of this manuscript.

    Regardless, this work provides an important insight into TMEM120 family and will serve as a basis for future investigations.

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