Fig 1.
Structure of EEA1 and the FYVE domain.
(A) Schematic of the full-length structure of dimeric EEA1, with the crystal structure of the FYVE domain dimer plus start of the coiled coil (PDB ID 1JOC) in yellow, followed by a ~200 nm coiled coil (dotted line) and a Rab5-binding domain. (B) The FYVE domains of the dimer, with four zinc ions (dark spheres) bound by 4-cysteine zinc fingers. Two bound inositol (1,3)-bisphosphate molecules (red/orange/green) are shown.
Fig 2.
(A) Set-up of the CG simulation, with the truncated FYVE dimer (yellow) placed at a minimum distance of 10 nm from the lipid bilayer (blue; orange PI(3)P molecules) and rotated at random. (B) Minimum distance for each of the 15 repeats, showing repeats without binding (grey, 7/15), with canonical binding (similar to the binding mode of the crystal structure, orange, 6/15) and with non-canonical binding (blue, 2/15). (C) Two snapshots from one of the repeats with canonical binding, after 800 and 911 ns. The snapshots show the hinge-mechanism by which FYVE binds to the membrane. Sketches below for clarity. (D) Potential of mean force (PMF) calculations for the binding of FYVE to a lipid membrane containing PC and PI(3)P, in the canonical binding mode, showing mean (dark blue line), standard deviation (shaded area) as well as minimum and maximum values (dotted lines). The PMF well depth is 67 ± 2 kJ/mol.
Fig 3.
Snapshots from a representative simulation at 0, 50, 100, 200, 300 and 500 ns. Heads 1 and 2 of the FYVE domain are highlighted (dotted circles) for the 100 ns snapshot. The inserted loop as seen at 100 ns shown in in green stick representation in the final panel.
Fig 4.
Atomistic simulations: distances, H-bonds and stalk tilt angle.
(A) Centre-of-mass distance between the membrane and each of the FYVE heads as function of time. (B) Number of H-bonds with PI(3)P headgroups for the FYVE heads. (C) Angle between the stalk axis and the bilayer normal as a function of time.
Fig 5.
Vesicle tethering and title of EAA1 coiled coil stalk relative to the bilayer normal.
The FYVE dimer is shown interacting with two PI(3)P molecules (orange) in a cell membrane (blue lines). Based on the analysis of our simulations this allows the coiled coil to tilt by up to 50° relative to the bilayer normal. Thus, the Rab5-binding domain (green) is able to search an area of ~0.09 μm2 for a contact to the target vesicle.