Structural insights into heterohexameric assembly of epilepsy-related ligand–receptor complex LGI1–ADAM22

Reviewer #1 (Public review):

The structure of a heterohexameric 3:3 LGI1-ADAM22 complex is resolved by Yamaguchi et al. It reveals the intermolecular LGI1 interactions and their role in bringing three ADAM22 molecules together. This may be relevant for the clustering of axonal Kv1 channels and control over their density. While it is currently not clear if the heterohexameric 3:3 LGI1-ADAM22 complex has a physiological role, the detailed structural information, presented here, allows us to pinpoint mutations or other strategies to probe the relevance of the 3:3 complex in future work.

The experimental work is done to a high standard, and I have no comments on that part. I do have several recommendations that I hope will be considered.

(1) A previously determined 2:2 heterodimeric complex of LGI1-ADAM22 was suggested to play a role in trans interactions. Could the authors discuss if the heterohexameric 3:3 LGI1-ADAM22 is more likely to represent a cis complex or a trans complex, or if both are possible?

(2) It is not entirely clear to me if the LGI1-ADAM22 complex is also crosslinked in the HS-AFM experiments. Could this be more clearly indicated? In addition, if this is the case, could an explanation be given about how the complex can still dissociate?

(3) The LGI1 and ADAM22 are of similar size. To me, this complicates the interpretation of dissociation of the complex in the HS-AFM data. How is the overinterpretation of this data prevented? In other words, what confidence do the authors have in the dissociation steps in the HS-AFM data?

(4) What is the "LGI1 collapse" mentioned in Figure 4c?

(5) Am I correct that the structure indicates that the trimerization is entirely organized by LGI1? This would suggest LGI1 trimerizes on its own. Can this be discussed? Has this been observed?

(6) C3 symmetry was not applied in the cryo-EM reconstruction of the heterohexameric 3:3 LGI1-ADAM22 complex. How much is the complex deviating from C3 symmetry? What interactions stabilize the specific trimeric conformation reconstructed here, compared to other trimeric conformations?

Reviewer #2 (Public review):

Summary:

The study by Yamaguchi et al. provides compelling evidence for the formation of a 3:3 complex between the ectodomain of ADAM22 and LGI1, as demonstrated using single-particle cryo-EM and HS-AFM. This represents the first instance in which the 3:3 complex has been resolved sufficiently to enable molecular modeling, allowing the authors to identify key interfaces mediating ADAM22-LGI1 interactions. HS-AFM revealed weak interactions within the 3:3 complexes, suggesting the dynamic nature of ADAM22-LGI1 interactions, which may play a role in modulating synaptic activity.

Strength:

A strength of this study lies in the novel identification of the 3:3 complexes, captured at an unprecedented level of resolution and validated by HS-AFM. This discovery, together with the authors' previous findings demonstrating a 2:2 stoichiometry, gives rise to an intriguing hypothesis regarding the dynamic nature of the ADAM22-LGI1 complex in regulating both cis- and trans-synaptic interactions.

Weakness:

The functional significance of these two complexes in the context of synapse remains speculative. Additionally, the structural presentations in Figures 1-3 (especially Figures 2-3) lack the clarity needed for general readers to fully understand the authors' key points. Enhancing the quality of these visual representations would greatly improve accessibility and comprehension.