Simulating the first steps of fertilization: Molecular Dynamics and Steered Molecular Dynamics studies of Juno and Izumo
Embargoed until 2026-03-06
Author
Date
2022Type
- Doctoral Thesis
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Abstract
Infertility affects approximately 15% of couples globally, amounting to 50 million of them being unable to conceive. Mostly, the cause for it remains unexplained. With assisted reproductive in vitro technologies, it is nowadays possible to overcome some of the shortcomings by injecting the sperm directly into the oocyte. However, the molecular details which lead to the fusion of sperm and oocyte, could help to apply less invasive treatments are still largely unknown. Sperm-egg adhesion and membrane fusion are the first molecular steps of sexual reproduction in mammals. The proteins that make the first physical contact between two plasma membranes of gametes comprise: Juno, on the outer membrane of the female egg, and Izumo, on the surface of the male sperm. Juno is a glycophosphatidylinositol (GPI)-anchored globular protein. Even though it is a member of the Folate Receptor (FR) family, it was shown not to bind folate when tested alone in a solution. Its feature distinguishing it from other FRs (and from Junos of other species), is an unusually long segment of its inhibitory loop of an unknown function. Izumo is a rod-like, trans-membrane protein with its extracellular parts composed of three segments: helical bundle on its N-terminal, hinge region and immunoglobulin (Ig)-like domain on its C-terminal. It can exist in two conformations, straight and boomerang, where its two domains are in a linear position or bent towards each other. Here, using computational tools, we studied the structure-function relationship of the Juno-Izumo complex. More specifically, we used approaches of equilibrium Molecular Dynamics (MD) and Steered Molecular Dynamics (SMD) simulations to gain structural insights into the trajectories by which tensile mechanical forces stretch and then dissociate the complex.
First, we took a closer look at Juno and Izumo, their structural states alone and in the complex with one another. We examined: how do their crystal structures change when getting hydrated in explicit water, which structural changes occur upon complexation, what are lifetimes of the bonds that form between Juno and Izumo and can this behavior explain the influence of some factors known to be present at fertilization. We could show that the Juno-Izumo contact site comprises a large cluster of more than 30 shortlived interactions. We then observed in silico, a spontaneous folate binding to Juno, while in complex with Izumo. However only if Juno was complexed to Izumo which stabilized the inhibitory loop positioned at the entrance of the folate binding pocket in an open position. Furthermore, we confirmed Izumo's preference for the “straight” state when in complex with Izumo and showed the possible co-existence of “straight” and “boomerang” conformations of Izumo in solution. Moreover, the MD data thus suggest that the presence of heavy metal ions (Zn²⁺) could shift the conformational distribution towards the “boomerang” shape. This behavior could potentially be disruptive for the Juno binding and therefore, could potentially explain structurally why exposure to heavy metals reduces fertility, or possibly be exploited to suppress fertilization. As sperm-egg fusion triggers the activation of the egg which includes the release of zinc ions, also referred to as “zinc spark”, into extracellular space, this increased concentration of Zn²⁺ could be, among its other roles, destined to prevent the binding of Izumo from subsequent sperms and thereby serve as the first, fast polyspermy block.
We then asked, given that Juno is GPI-anchored to the lipid membrane of the egg, how does it orient on the lipid membrane and to what extent does this depend on the lipid composition. Using MD on different length and resolution scales, Coarse Grained and atomistic, we showed preferred Juno poses on the membrane and its possible lipid interaction sites. The preferred orientation on a lipid membrane that mimics the composition of lipid rafts, suggests that Juno can form dimers and linear multimers, and that this is potentially enhanced via loss of its intramolecular disulfide bonds. This local organization of Juno on the egg membrane could be another regulatory mechanism, tightly coordinating the process of sperm-egg adhesion and fusion.
Finally, we asked how could the mechanical forces, undoubtedly present at the sperm-egg adhesion site, impact the Juno-Izumo interaction. To this end we have created a series of constant pulling SMD simulations. To assess its unbinding pathway, we chose orientations that could be easily reproduced in wet lab experiments to pull two proteins apart. We have observed an initial increase in the contact area in response to the pulling force, different trajectories towards the rupture, one of which showed an extended lifetime and multi-branched force propagation pathways across the complex enabling distribution of the pulling forces. Taken together, those results suggest that the Juno-Izumo complex has high mechanical stability, with some characteristics of catch bond-like behavior. Further, to understand Juno-Izumo response to shear stress, we pulled on the complex in 5 other directions and observed alternative behavior of the Juno-Izumo complex. As sperms from different species push with different velocities, the mechanical design of the Juno-Izumo complex could therefore have evolved to serve as a first force-controlled species selection, suggesting that the mechanical stability of the complex depends on the direction from which the force is applied.
Many important factors, present at the fertilization are not accessible via experimental methods of structural biology, particularly if they are only transiently present in the gametes adhesion and fusion process, such as partial restraints posed by membrane binding or mechanical forces coming from the energetic agellum movement. Using MD and SMD simulations, we were able to provide new insights into the interaction of Juno-Izumo complex with their additional binding that could be of a significant clinical relevance. The in silico results will help to decipher the mechanical design of the Juno-Izumo complex, important for the early stages of sexual reproduction. Our results possess great potential to inspire new experimental studies and hopefully, develop new infertility treatments and diagnosis strategies. Show more
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https://doi.org/10.3929/ethz-b-000601877Publication status
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ETH ZurichOrganisational unit
03640 - Vogel, Viola / Vogel, Viola
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Is supplemented by: https://doi.org/10.3929/ethz-b-000601882
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