Membrane localization and dynamics of geranylgeranylated Rab5 hypervariable region.

The small GTPase Rab5 is a key regulator of endosomal trafficking processes and a marker for the early endosome. The C-terminal hypervariable region (HVR) of Rab5 is post-translationally modified at residues Cys and Cys to accommodate two geranylgeranyl anchors (C20 carbon chain length) in order to associate Rab5 with the membrane. The structural role of the HVR regarding protein-early endosome membrane recruitment is not resolved due to its high degree of flexibility and lack of crystallographic information. Here, full-atomistic and coarse-grained molecular dynamics simulations of the truncated Rab5 HVR in three model membranes of increasing complexity (pure phospholipid bilayer, ternary membrane with cholesterol, six-component early endosome) were performed. Specific electrostatic interactions between the HVR Arg residue and the phosphate group of the inositol ring of PI(3)P were detected. This shows that PI(3)P acts as a first contact site of protein recruitment to the early endosome. The free energy change of HVR extraction from the bilayer was largest for the physiological negatively charged membrane. 5μs coarse-grained simulations revealed an active recruitment of PI(3)P to the HVR supporting the formation of Rab5- and PI(3)P enriched signaling platforms.