Charge reversal in predicted C-terminal α-helix activates ATL2. (A) AlphaFold structure prediction (Jumper et al., 2021) of the ATL2 tail rendered in cartoon form in PyMOL. Nonpolar residues of the amphipathic helix shown as sticks and highlighted in orange. Inhibitory domain residues selected for charge reversal also shown as sticks and highlighted in red (acidic) or blue (basic). Charge reversal variants tested in B and initial fusion rates for each variant are indicated, and residues substituted in each variant are underlined. Also shown is a rotated view of the helix to highlight the slight bend in the helix predicted by AlphaFold, residues P548 and G550 that may mediate the bend highlighted as spheres, and the P548A/G550A variant designed as a possible test of the role of the predicted helical bend. AH, amphipathic helix. (B) Lipid mixing by each of the indicated variants in A. All proteins were incorporated at a 1:1,000 protein/lipid ratio, and the data are the average of at least two independent traces.