Abstract
HAPLESS2 (HAP2) is a broadly conserved, gamete-expressed transmembrane protein that was shown recently to be structurally homologous to viral class II fusion proteins, which initiate fusion with host cells via insertion of fusion loops into the host membrane. However, the functional conformation of the HAP2 fusion loops has remained unknown, as the reported X-ray structure of Chlamydomonas reinhardtii HAP2 lacked this critical region. Here, we report a structure-guided alignment that reveals diversification of the proposed HAP2 fusion loops. Representative crystal structures show that in flowering plants, HAP2 has a single prominent fusion loop projecting an amphipathic helix at its apex, while in trypanosomes, three small nonpolar loops of HAP2 are poised to interact with the target membrane. A detailed structure-function analysis of the Arabidopsis HAP2 amphipathic fusion helix defines key residues that are essential for membrane insertion and for gamete fusion. Our study suggests that HAP2 may have evolved multiple modes of membrane insertion to accommodate the diversity of membrane environments it has encountered during eukaryotic evolution.
Highlights
The fusion of gamete plasma membranes to form a zygote is central to sexual reproduction, yet a molecular mechanism for this fundamental process has only very recently been proposed
The domain II tip is the region of class II fusion proteins that must firmly insert into target membranes to initiate the fusion process
We found that the soluble, wild-type (WT) Arabidopsis thaliana HAPLESS 2 ectodomain (AtHAP2e) migrated to the top of the gradient, in contrast to a triple alanine substitution mutant in αF, which remained in the bottom of the tube (Fig 5A, S1 Data), indicating that HAPLESS 2 (HAP2) binds membranes and that the bulky nonpolar side chains of αF are required
Summary
The fusion of gamete plasma membranes to form a zygote is central to sexual reproduction, yet a molecular mechanism for this fundamental process has only very recently been proposed. The crystal structure of the C. reinhardtii HAPLESS 2 (HAP2) ectodomain (CrHAP2e) [1] revealed that this broadly conserved, gamete-expressed transmembrane protein [2,3,4] has the same three-dimensional fold as class II viral fusion proteins. Like its viral counterparts, HAP2 initiates gamete fusion by insertion of fusion loops into the opposing gamete plasma membrane [5]. HAP2 is homologous to the viral class II fusion proteins, which fold into an articulated rod made of three β-sheet-rich domains, termed I, II, and III, with the central domain I flanked by domains II and III in the prefusion form [7,8,9]. Domain III connects to the C-terminal transmembrane domain via a flexible linker, while domain II bears the fusion loops at the opposite end
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