Structural studies of cellular membrane fusion proteins involved in C. Elegans organogenesis – CellMemFusion

Essential processes in developmental biology, such as embryogenesis or organogenesis, rely in specific and tightly controlled cell-cell fusion events. Events like sperm-egg fusion for fertilization, myoblast fusion to form myotubes in skeletal muscle formation, monocyte fusion to form osteoclasts in bone homeostasis, etc., are just a few examples.
In contrast to the intracellular fusion events involving SNARE proteins, or to virus-cell fusion during infection of a cell, little is known about the proteins that are responsible for inducing the cell-cell fusion events during development.

Structural studies have been extremely useful in the case of SNAREs and of viral envelope proteins to identify their fusogenic mechanism. In this project, we propose to determine the crystal structure of two cell-cell fusion proteins – the only ones that have been clearly identified to date – proteins EFF-1 and AFF-1 which belong to the FF family of fusion proteins of the nematode C. Elegans. The fusion event controlled by the FF proteins occurs in organogenesis during development of the nematode, and is essential for the formation of the epidermis and of the vulval ring.

Knowledge of these structures will constitute a major advance in the field, giving it a considerable push forward. The resutls will benefit the whole community of developmental biologists, as well as the biophysicists studying the mechanism of lipid pore formation induced by proteins.

The novelty of this proeject relates to the fact that no cell-cell fusogen has been characterized structurally to date, in spite of the importance of cell-cell fusion processes in developmental biology. Comparative studies have been extremely informative in the case of viruses, where they allowed to extract the common features present in proteins that have a completely different 3D fold, like the members of the different structural classes, yet they all adopt a similar hairpin conformation to induce membrane fusion. Such information helped to immediately interpret structure of the SNARE oligomer, in which the segments anchored in target and vesicle membranes also appear at the same end of a very stable protein rod. We anticipate that knowing the 3D structures of the FF protein will produce its lot of new surprises and reveal important information to understand their function. The fact that these proteins only work to induce homotypic cell-cell fusion is a very interesting difference to all the other cases studied so far. Furthermore, the recognition of a domain folded as a growth receptor hormone, introduces a new twist that we expect the structure will help clarify.

In preliminary work, we have obtained small crystals of EFF-1, which will require quite an effort to optimize until the diifrraction quality is enough to determine the 3D structure. The best diffraction obtained so far is about 4Å resolution, which is not enough to accurate determine the structure. We have, in addition, identified a few heavy atom binders, which will be used for phasing as soon as the diffraction is sufficient. To achieve this goal, we will screen homologs of the EFF-1 protein from the varioaus other nematodes for which the sequnces are available. We will also sub-contract to anexternal company to obtain monoclonal antibodies against EFF-1, in order to generate Fab fragments to use for co-crystallization. In the case of glycoproteins such as thisone, crystals of such complexes often diffract better than the protein alone. We anticipate that one of these approaches will provide crystals with sufficient diffraction quality to succesfully detemrine the 3D structure.