Structural insights into heterohexameric assembly of epilepsy-related ligand–receptor complex LGI1–ADAM22
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eLife Assessment
This is a useful follow-up on previous work on the same LGI1-ADAM22 complex using cross-linking to stabilize a trimeric state that the authors had previously observed by SEC-MALS and small-angle X-ray scattering (the previous crystal structure was determined in a dimeric form). A strength of this solid work is that oligomeric states do not affect the critical interaction between LGI1 and ADAM23, so the previous conclusions are still valid. A weakness is that the physiological relevance of the trimeric assembly is unclear.
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Abstract
Leucine-rich glioma-inactivated 1 protein (LGI1) is a secreted neuronal protein consisting of the N-terminal leucine-rich repeat (LRR) and C-terminal epitempin repeat (EPTP) domains. LGI1 is linked to epilepsy, a neurological disorder that can be caused by genetic mutations of genes regulating neuronal excitability ( e . g ., voltage- or ligand-gated ion channels). ADAM22 is a membrane receptor that binds to LGI1 extracellularly and interacts with AMPA-type glutamate receptors via PSD-95 intracellularly to maintain normal synaptic signal transmission. Structural analysis of the LGI1–ADAM22 complex is important for understanding the molecular mechanism of epileptogenesis and developing new therapies against epilepsy. We previously reported the crystal structure of a 2:2 complex consisting of two molecules of LGI1 and two molecules of the ADAM22 ectodomain (ECD), which is suggested to bridge neurons across the synaptic cleft. On the other hand, multiangle light scattering, small-angle X-ray scattering, and cryo-EM analyses have suggested the existence of a 3:3 complex consisting of three molecules of LGI1 and three molecules of ADAM22. In the previous cryo-EM analysis, many observed particles were in a dissociated state, making it difficult to determine the three-dimensional (3D) structure of the 3:3 complex. In this study, we stabilized the 3:3 LGI1–ADAM22 ECD complex using chemical crosslinking and determined the cryo-EM structures of the LGI1 LRR –LGI1 EPTP – ADAM22 ECD and 3:3 LGI1–ADAM22 ECD complexes at 2.78 Å and 3.79 Å resolutions, respectively. Furthermore, high-speed atomic force microscopy (HS-AFM) visualized the structural features and flexibility of the 3:3 LGI1–ADAM22 ECD complex in solution. We discuss new insights into the interaction modes of the LGI1–ADAM22 higher-order complex and the structural properties of the 3:3 LGI1–ADAM22 complex.
Significance
The neuronal secretory protein Leucine-rich glioma-inactivated 1 (LGI1) and its receptor protein ADAM22 play a critical role in maintaining normal synaptic signal transmission. Genetic mutations in LGI1 are linked to epilepsy. Structural analysis of the LGI1–ADAM22 complex is crucial for understanding the molecular mechanisms of epileptogenesis and for developing targeted treatments. In this study, we determined the cryo-EM structure of the heterohexameric LGI1–ADAM22 complex and visualized the dynamics of this complex by high-speed atomic force microscopy.
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eLife Assessment
This is a useful follow-up on previous work on the same LGI1-ADAM22 complex using cross-linking to stabilize a trimeric state that the authors had previously observed by SEC-MALS and small-angle X-ray scattering (the previous crystal structure was determined in a dimeric form). A strength of this solid work is that oligomeric states do not affect the critical interaction between LGI1 and ADAM23, so the previous conclusions are still valid. A weakness is that the physiological relevance of the trimeric assembly is unclear.
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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 …
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?
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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 …
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.
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