ABSTRACT
The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.
Subject(s)
Diterpenes/chemistry , Guanine Nucleotide Dissociation Inhibitors/chemistry , Molecular Dynamics Simulation , Multiprotein Complexes , Protein Prenylation , rab5 GTP-Binding Proteins/chemistry , Animals , Cattle , Diterpenes/metabolism , Guanine Nucleotide Dissociation Inhibitors/genetics , Guanine Nucleotide Dissociation Inhibitors/metabolism , Humans , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolismABSTRACT
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 Cys212 and Cys213 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 HVR206-215 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 HVR206-215 Arg209 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 HVR206-215 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 HVR206-215 supporting the formation of Rab5- and PI(3)P enriched signaling platforms.
Subject(s)
Cholesterol/chemistry , Diterpenes/chemistry , Lipid Bilayers/chemistry , Phosphatidylinositol Phosphates/chemistry , rab5 GTP-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites , Cholesterol/metabolism , Diterpenes/metabolism , Endosomes/chemistry , Endosomes/metabolism , Glycosylation , Humans , Lipid Bilayers/metabolism , Models, Molecular , Molecular Dynamics Simulation , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Signal Transduction , Sphingomyelins/chemistry , Sphingomyelins/metabolism , Thermodynamics , rab5 GTP-Binding Proteins/metabolismABSTRACT
Rab5 is a small GTPase and a key regulator in early endosomal trafficking. Rab5 and its effectors are involved in a large number of infectious diseases and certain types of cancer. We performed µs atomistic molecular dynamics simulations of inactive and active full-length Rab5 anchored to a complex model bilayer with composition of the early endosome membrane. Direct interactions between the Rab5 G domain and the bilayer were observed. We found two dominant nucleotide-dependent orientations characterised by a different accessibility of the switch regions. The "buried switch" orientation was mainly associated with inactive Rab5 accompanied with a rather extended structure of the hypervariable C-terminal region. Active Rab5 preferred an orientation in which the switch regions are accessible to effector proteins. These structural differences may provide an opportunity to selectively target one Rab5 state and lead to new approaches in the development of Rab5-specific therapies.
