Reviewer #2 (Public Review):
In the manuscript entitled "The Crystal Structure of Bromide-bound GtACR1 Reveals a Pre-activated State in the Transmembrane Anion Tunnel", Li et al. analyzed the effect of bromide binding to GtACR1 by X-ray crystallography and electrophysiology. The authors propose that a bromide ion is bound to the intracellular pocket in the dark, inactivated state and induces a structural transition from an inactivated to a pre-activated state.
I agree that some of the amino acid residues in the current crystal structure change their conformations compared to the previous one reported in 2019 (Li et al., 2019), and it is very impressive that the authors determined the structure using state-of-the-art crystallography technique, ISIMX. However, unfortunately, most of the conclusions and claims described in the manuscript are not well supported by the authors' data.
1) The most serious problem is that the evidence of bromide binding is too weak. The authors showed the composite omit map in Supplementary Figure 1A, but they should present an anomalous difference Fourier map to validate the bromide binding. The authors also claim that they replaced the bromide ion to the water, run the PHENIX refinement, and observed a strong positive electron density at the bromide position in the Fo-Fc difference map (Supplementary Figure 1B). However, when I do the same thing using the provided coordinate and map (I really appreciate the honesty and transparency of the authors), I could not reproduce their result; a weak positive electron density is observed between the bromide position and Pro58 in chain A and there is no positive peak at the position in chain B (Fo-Fc, contoured at 3σ). I am wondering the occupancy and B-factor of the water molecule they show in Supplementary Figure 1B.
In addition to the insufficient evidence, the current models of bromide ions have significant steric clashes. The PDB validation report shows that the top 5 serious steric clashes observed in the coordinate are the contacts between the bromide ions and surrounding residues (PDB validation report, Page 10). I analyzed them and found that the distance between the bromide ion and CG and CD atoms of Pro58 in chain A are only 2.43Å and 2.36Å, respectively. The authors claim that such a close proline-halide interaction has also been observed in the structure of the chloride-pump rhodopsin CIR, but in the structure (PDB ID: 5G28), the distances between the chloride ion and CD and CG atoms of Pro45 are much larger (3.43 and 3.91Å, respectively) and there is no steric clash. Moreover, the authors claim that Pro58 changes its conformation by bromide binding, but it is very possible that the PHENIX program just displaces Pro58 to alleviate the steric clash between the proline and the bromide ion, so the authors should carefully check the possibility.
Overall, the authors should analyze the density again, provide more solid evidence for the bromide binding such as anomalous difference Fourier map, and if they could, they should correct the current significant steric clashes in their models.
2) To analyze the functional importance of putative bromide binding, the authors prepared W246E and W250E mutants and analyzed their electrophysiological properties. Because tryptophan and glutamate are so different in terms of volume and charge, they should analyze other mutants as well. The authors claim that bromide is stabilized by a hydrogen bond interaction formed by the indole NH group of W246, so they should at least test the W246F mutant.
3) The authors claim that the bromide binding in the intracellular pocket induces the conformational change of R94, but the causal relationship is doubtful. As mentioned in the manuscript, R94 forms a salt-bridge with D234 in chain A. However, the arginine has a completely different conformation and does not have any interaction with D234 in chain B. If the bromide binds both in chain A and B and induces the conformational change of R94, why only R94 in chain A interacts with D234? The authors change the pH in the crystallization condition compared to their 2019 study (Li et al., 2019), so the pH may affect the protonation state of D223 and/or other titratable residues and induces the conformational change of R94. The authors should provide more solid evidence for the causal relationship between the bromide binding and the conformational change of R94.
4) The authors assume that the conformational change of R94 creates a functional anion binding site with the Schiff base in GtACR1, but it is too speculative. If the anomalous difference Fourier map does not support the idea, they should delete it.