Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse

Reviewer #1 (Public Review):

Summary:

Taking advantage of the Alphafold-multimer program, which predicts the tertiary structure of the macromolecular complex, the authors analyzed the interaction of essential factors involved in sperm-egg fusion. In particular, the authors predicted that the presence of a large complex of the novel factor TMEM81 with IZUMO1, SPACA6, JUNO, and CD9.

Strengths:

The authors postulated that the type I transmembrane sperm protein TMEM81 may be involved in gamete fusion, as predicted by the Alphafold-multimer.

Weaknesses:

All data except Figure 1 are mere predictive models, and their physiological importance is extremely unreliable. In addition, the data lacks experimental validation compared to another group's preprint (https://www.biorxiv.org/content/10.1101/2023.07.27.550750v1).

Reviewer #2 (Public Review):

Summary:

Fertilization is a crucial event in sexual reproduction, but the molecular mechanisms underlying egg-sperm fusion remain elusive. Elofsson et al. used AlphaFold to explore possible synapse-like assemblies between sperm and egg membrane proteins during fertilization. Using a systematic search of protein-protein interactions, the authors proposed a pentameric complex of three sperm (IZUMO1, SPACA6, and TMEM81) and two egg (JUNO and CD9) proteins, providing a new structural model to be used in future structure-function studies.

Strengths:

1. The study uses the AlphaFold algorithm to predict higher-order assemblies. This approach could offer insights into a highly transient protein complex, which is challenging to detect experimentally.
2. The article predicts a pentameric complex between proteins involved in fertilization, shedding light on the architectural aspects of the egg-sperm fusion synapse.

Weaknesses:

1. The procedures and discriminator scores used to evaluate specific from non-specific complexes were developed previously by the same authors. Therefore, in this manuscript, they are not contributing a new method.
2. The proposed model, which is a prediction from a modeling algorithm, lacks experimental validation of the identity of the components and the predicted contacts.

It is noteworthy that in an independent study, Deneke et al. provide experimental evidence of the interaction between IZUMO1/SPACA6/TMEM81 in zebrafish. This is an important element that supports the findings presented in this manuscript.

Reviewer #3 (Public Review):

Summary:

Sperm-egg fusion is a critical step in successful fertilization. Although several proteins have been identified in mammals that are required for sperm-egg adhesion and fusion, it is still unclear whether there are other proteins involved in this process and how the reported proteins complex and/or cooperate to complete the fusion process. In this study, the authors first identified TMEM81 as a structural homologue of IZUMO1 and SPACA6, and using AlphaFold-Multimer, a recent advance in protein complex structure prediction, predicted the interactions between human proteins associated with gamete fusion. While the prediction is compelling and well discussed, the experimental evidence to verify this interaction is lacking, so the prediction remains a hypothesis.

Strengths:

The authors present a pentameric complex formation of four previously reported proteins involved in egg/sperm interaction together with TMEM181 using a deep learning tool, AlphaFold-Multimer.

Weaknesses:

While it is intriguing to see that some of the proteins involved in sperm-egg interaction are successfully predicted to be assembled into a single multimeric structure by AlphaFold-Multimer, it is necessary to experimentally validate the interactions. As there are more candidate proteins in the process, it will be necessary to test other possible protein interactions to prove the adequacy of the candidates chosen by the authors, as similar analysis with some other proteins will provide more rationale for further 3D multi-protein modeling. In addition, the lack of biochemical data to support the predicted bindings between proteins limits the proposed complex to remain mainly hypothetical.