Avalilação PREreview de Cryo-EM structures of insect IRs reveal the structural adaptations of a glutamate receptor ancestor for environmental sensing

Overall assessment

Petrovich et al. investigated how insect ionotropic receptors (IRs) adapted an ancestral glutamate-receptor architecture to detect different environmental signals. The authors relied mainly on cryo-EM, together with sequence analysis and phylogenetic comparisons, to characterize three IR complexes: IR8a/IR75b, IR25a/IR76b, and IR25a/IR93a/IR40a. The structures reveal an interlocking helical beam formed by the primary co-receptors that organizes the complex and separates the tuning subunits. The authors also identified structural features that may contribute to subunit assembly specificity and proposed how interactions between tuning subunits and co-receptors could coordinate channel gating. These findings provide a structural basis for understanding how conserved co-receptors assemble with diverse sensory subunits.

Strengths

The cryo-EM structures provide strong evidence for the subunit arrangements and the interlocking co-receptor beam that organizes IR complexes. Comparing the three complexes helps explain how conserved co-receptors assemble with different sensory subunits. These findings improve our understanding of how an ancestral glutamate-receptor architecture was adapted for environmental sensing.

Weaknesses

The structural comparisons in Figures 6–7 identify features associated with co-receptor specificity. However, as the authors acknowledge, these features may also reflect shared ancestry, and their role in determining assembly remains uncertain. Making this limitation clearer in the abstract and Results would help readers understand the scope of the proposed assembly rules.

In addition, the IR25a/IR93a/IR40a complex contains subunits from three different species, and the IR25a/IR76b structure lacks the tuning subunit IR41a. The authors might consider discussing how these differences could affect the interpretation of the results, particularly whether the observed subunit arrangements and interactions represent those in native receptors.

Major Comments

The cryo-EM structures provide convincing evidence for the overall architecture of IR complexes. However, the proposed roles of the beam in assembly (Figures 1C and 5) and the coupling interfaces in gating (Figures 4B and 5D) have not been directly tested. The authors might consider mutating key interface residues and examining complex formation by co-purification and size-exclusion chromatography. Where reliable recordings are possible, electrophysiology with cell-surface expression controls could help test the proposed gating mechanism. These experiments would strengthen the connection between the structural findings and receptor function.

Minor Comments

1. The structural comparisons in Figures 6–7 identify features that may explain co-receptor specificity. However, these features could also reflect shared evolutionary history, as the authors acknowledge in the limitations section. The authors might consider briefly noting this possibility in the Results when presenting the proposed assembly rules, so readers can better understand the limits of these predictions.

2. The interactions shown in Figures 4B and 5D suggest a gating mechanism, but their functional roles remain untested. The authors might consider testing interface mutations by electrophysiology, with cell-surface expression controls to distinguish gating effects from trafficking defects. If reliable recordings remain difficult, the mechanism could be presented more clearly as a hypothesis in the Discussion.

3. Figure 5 would benefit from a simple schematic identifying the primary co-receptors, secondary co-receptors, and tuning subunits in each complex. Including their species of origin and stoichiometry would help readers follow the comparisons between these assemblies.

4. The construct-design section states that the last 49 amino acids of IR8a were deleted. The authors might briefly explain whether this truncation improved expression or stability and note any known role of the deleted region. This would help readers understand why this construct was selected and assess how the truncation might affect the interpretation of the structure.

I reviewed this preprint as part of the Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.