Fig. 3: Loose crossover dimer formation and the linker region between the 3HB and TMD play roles in ATL-mediated membrane fusion.

a Ribbon representation of the crystal structure of ATL1_cyto dimer in Form 2 (PDB code 3QNU) conformation rendered in PyMOL. The molecules are shown as in Fig. 1a. S398 is represented as a purple stick in one protomer. b, c As in Fig. 2c, d, but with ATL1_cyto-K-S398Y. d Relative occupancy of the low-FRET state and high-FRET state for ATL1_cyto-K, ATL1_cyto-K-S398Y, ATL1_cyto-K-N440T, and ATL1_cyto-K-H443P dimers in the presence of GTPγS as derived from histograms. The data from Fig. 2e for GTPγS-bound ATL1cyto-K dimer are shown for comparison. Data are presented as mean ± SD (n = 3 independent experiments), determined from three randomly assigned populations of all FRET traces. ns, not significant; *P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. P-values: 0.0441 (ATL1_cyto-K vs ATL1_cyto-K-S398Y), 0.9995 (ATL1_cyto-K vs ATL1_cyto-K-N440T), 0.6430 (ATL1_cyto-K vs ATL1_cyto-K-H443P). e Relative free energy models of ATL1_cyto-K and ATL1_cyto-K-S398Y in the presence of GTPγS. The differences between the low-FRET state and high-FRET state are roughly scaled and marked below the corresponding states. f Ribbon representations of the crystal structures of ATL1_cyto (top, PDB code 4IDN) and ATL1_cyto-N440T (bottom, PDB code 4IDP) dimer in Form 3 conformation rendered in PyMOL. The molecules are shown as in Fig. 1a. The linker regions between the 3HBs and TMDs are colored in brown. The N440 (N440T) and H443 are represented as green and cyan sticks, respectively. g, h As in Fig. 2c, d, but with ATL1_cyto-K-N440T. i Time courses of membrane fusion between donor and acceptor proteoliposomes containing WT dmATL, dmATL K418P (H443P in human ATL1), or no protein in the presence of GTP as assessed by dequenching of NBD-PE fluorescence (mean ± SD, n = 3 independent experiments). j, k As in Fig. 2c, d, but with ATL1_cyto-K-H443P. Source data are provided as a Source Data file.