Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations

Author summary Peripheral proteins bind to the surfaces of specific membranes within eukaryotic cells and thereby coordinate dynamic interactions between sub-cellular compartments. The FYVE domain of EEA1 recognizes the membranes of endosomes by binding to the anionic headgroup of a specific lipid, phosphatidylinositol-3-phosphate (PI(3)P), which is present in those membranes. We use molecular dynamics simulations at two different resolutions (coarse-grained and all atom) to define the mechanism whereby a dimeric FYVE domain binds to model membranes containing PI(3)P. Hinge-bending flexibility between the bound FYVE domain and the coiled coil stalk to which it is attached is observed. In the intact EEA1 molecule the coiled coil is 200 nm long. This 'amplifies' the flexibility observed at the FYVE hinge end such that the other end of the EAA1 protein can sweep out an area of about 0.1 mu m(2) in its search for a second endosome with which to interact. In this way EAA1 acts as a long stalk with two 'sticky' ends to help draw together two specific membrane surfaces. Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50-60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer c