Abstract
HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of Chlamydomonas HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on Chlamydomonas gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.
Highlights
Sexual reproduction in eukaryotes is key to evolution of organismal complexity and diversity
We interpret our structural and functional results, and structures of viral fusogens such as those shown in Figures 5 and 6, in support of a model in which the fusion loops in each HAP2 monomer approach the 3-fold axis in the fusion state interrogated in vivo by mutation
We have characterized the crystal structure of HAP2 from Chlamydomonas reinhardtii in a trimeric fusion state at 2.6 A, the dynamics of its polypeptide backbone in both monomeric and trimeric states, and the functional importance in gamete fusion of residues in its fusion loop
Summary
Sexual reproduction in eukaryotes is key to evolution of organismal complexity and diversity. In the final steps of conformational change, which are yet to be resolved structurally, the remainder of the stem containing hydrophobic elements packs against the trimer and the target membrane, and drags the transmembrane domain anchored to the viral membrane into intimate contact with the fusion loops moored in the target endosomal membrane. Mutational analyses show that the a1 and a2 helices, which are parallel to one another, have a much more critical function in fusion than the h1 helix, which is tangential to and more distant from the central axis These results, the large amount of splaying of the fusion loop of each protomer from the trimer central axis, and hydrogen-deuterium exchange (HDX) results showing flexibility within D2 suggest that the crystallized form of HAP2 is an intermediate in the structural transition pathway for membrane fusion. We propose that during the final stages of fusion, the apically localized fusion loops of HAP2 tilt towards the trimer core to form a more compact fusion surface
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