Final Report Summary - CELCELFUS (Cell-Cell fusion in fertilization and developmental biology: a structural biology approach)
Intracellular membrane fusion events – in particular, SNARE-meditated fusion of internal vesicles with organelles or with the plasma membrane, have been substantially studied throughout the years. Similarly, the study of enveloped viruses and the way they invade their host cells has provided important insight into virus-cell fusion mechanisms. In stark contrast, at the beginning of this ERC grant extremely little was known about cell-cell fusion events. These processes are, however, essential for organ development in multicellular organisms, as well as for the sexual reproduction of eukaryotes, which involves fusion of male and female gametes. In this context, the determination of the X-ray structure of HAP2 has been a founding moment – a moment in which it became clear that the protein responsible for the fusion of gametes in members of four out of the five eukaryotic kingdoms (except for fungi) – is a distant ortholog of viral envelope proteins driving membrane fusion. Indeed, their three-dimensional (3D) fold shows that they have the same sequential arrangement of secondary structure elements (SSE) along their primary sequence, the same clustering of these SSE to form domains, (i.e. same tertiary structure), and the same quaternary organization in their lowest-energy, post-membrane fusion trimeric conformation. Because these proteins display no detectable amino acid sequence similarity, the homology was impossible to detect by sequence comparisons. Yet the probability that two proteins with different origins converge during their evolution into an identical - and quite complex - 3D fold is extremely low - essentially null. Already within viruses, it had come as a surprise that apparently unrelated viruses displayed envelope proteins with the same 3D fold. The study of virus fusion had also shown that proteins with unrelated folds are capable of executing the membrane fusion reaction, leading to their classification into three structural classes, I, II and III, highlighting the fact that their common function as membrane fusogens is not necessarily related to a particular 3D fold. The most likely explanation for the similarity observed between HAP2 and the viral proteins is that they derive from the same ancestral protein whose primary sequence has evolved to the point that sequence similarity is now undetectable. This ancestral protein is the postulated precursor of the envelope proteins of extant class II viruses, of HAP2, as well as the EFF-1 protein required for skin formation in nematodes (EFF-1 is fusion protein also folded as a class II viral fusion protein, identified as such by my group previous to this grant - a discovery that led to our proposal to study HAP2 within the Celcelfus grant).
The viral class II membrane fusion proteins occur in RNA viruses displaying a regular lattice of surface glycoproteins. They have the capacity of controlling membrane curvature and drive the process of budding of closed viral particles. Their organization at the surface of virus particles keeps them trapped in a metastable state. Only when they are incorporated into their host cell by endocytosis, they are triggered by the acidic environment of the endosome to undergo a fusogenic conformational transition. The end point of this transition is their trimeric post-fusion state - the same state in which we characterized HAP2 in our structural studies. Very recently, we have identified a conformation of HAP2 that is different, which likely corresponds to its pre-fusion form. Our results now open the way to understand the details of the fusogenic conformational change of HAP2, and also how HAP2 is organized at the cell surface prior to fusion. The results from the Celcelfus grant have now put us in a situation of exploring HAP2 function - inspired from the mechanism of action of their viral counterparts - that we could not have suspected five years ago, when applying for the grant.
The viral class II membrane fusion proteins occur in RNA viruses displaying a regular lattice of surface glycoproteins. They have the capacity of controlling membrane curvature and drive the process of budding of closed viral particles. Their organization at the surface of virus particles keeps them trapped in a metastable state. Only when they are incorporated into their host cell by endocytosis, they are triggered by the acidic environment of the endosome to undergo a fusogenic conformational transition. The end point of this transition is their trimeric post-fusion state - the same state in which we characterized HAP2 in our structural studies. Very recently, we have identified a conformation of HAP2 that is different, which likely corresponds to its pre-fusion form. Our results now open the way to understand the details of the fusogenic conformational change of HAP2, and also how HAP2 is organized at the cell surface prior to fusion. The results from the Celcelfus grant have now put us in a situation of exploring HAP2 function - inspired from the mechanism of action of their viral counterparts - that we could not have suspected five years ago, when applying for the grant.