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. 2019 Sep 26;20(19):4773.
doi: 10.3390/ijms20194773.

Structure and Dynamics of Mono- vs. Doubly Lipidated Rab5 in Membranes

Eileen Münzberg  1 , Matthias Stein  2
Affiliations

Structure and Dynamics of Mono- vs. Doubly Lipidated Rab5 in Membranes

Eileen Münzberg et al. Int J Mol Sci. .

Abstract

The Rab5 small GTPase is a regulator of endosomal trafficking and vesicle fusion. It possesses two adjacent cysteine residues for post-translational geranylgeranylation at its C-terminus for the protein to associate with the early endosome membrane. We compare the effect of mono-lipidification of only one cysteine residue with the doubly modified, fully functional Rab protein in both guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states and in different membranes (one, three, and six-component membranes). Molecular simulations show that the mono-geranylgeranylated protein is less strongly associated with the membranes and diffuses faster than the doubly lipidated protein. The geranylgeranyl anchor membrane insertion depth is smaller and the protein-membrane distance distribution is broad and uncharacteristic for the membrane composition. The mono-geranylgeranylated protein reveals an unspecific association with the membrane and an orientation at the membrane that does not allow a nucleotide-specific recruitment of further effector proteins. This work shows that double-lipidification is critical for Rab5 to perform its physiological function and mono-geranylgeranylation renders it membrane-associated but non-functional.

Keywords: GTPase; diffusion; lipid bilayer; molecular dynamics; post-translation modification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rab proteins shuttle between inactive, GDP-bound, and active, guanosine triphosphate (GTP)-bound states, which affect their cellular localization. The protein is post-translationally modified and lipidated by a transferase with two 20-carbon lipophilic geranylgeranyl isoprene units. Transport between different vesicular membranes is realized by RabGDI, which exclusively binds Rab(GDP). Guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP to GTP and thereby the activation of Rab proteins. Rab(GTP) is solely membrane-associated and able to recruit a large number of effector proteins. GTP hydrolysis and recovery of Rab(GDP) is achieved by GTPase activating proteins (GAPs) [5].
Figure 2
Figure 2
Amino acid sequence of the Rab5 protein and important functional regions. From the 215 residues, only the catalytic G domain structure from Gly15–Glu185 is experimentally resolved. The post-translationally attached geranylgeranyl chains are located at C-terminal residues Cys212 and Cys213 (marked with arrows).
Figure 3
Figure 3
Variation of the average acyl chain order parameter in vicinity to the GG anchor (in a cutoff radius = 0.5 nm) from the global average in the three investigated membrane models for different protein systems.
Figure 4
Figure 4
Distribution of the GG anchor insertion depths of (A) double-GG Rab5 and (B) mono-GG Rab5 in different membranes.
Figure 5
Figure 5
The z-distances between each amino acid residue and the membrane surface were monitored over the molecular dynamics (MD) trajectories. Left: (GG,GG)-Rab5 GDP and GTP; right: (GG)-Rab GDP and GTP.( A) in pure POPC, (B) the ternary mixture, and (C) the six-component membrane.
Figure 6
Figure 6
Comparison of mono-GG and double-GG Rab5 structural parameters in the six-component membrane with regard to (A) the RMSF, (B) the protein radius of gyration, and (C) the internal dihedral angle ω as a function of the G domain orientation θ.
Figure 7
Figure 7
Final conformations of A) mono-GG Rab5(GDP) and B) mono-GG Rab5(GTP) in the physiological early endosomal six-component membrane.
Figure 8
Figure 8
Initial conformations of full-length membrane-bound (A) Rab5(GDP) and (B) Rab5(GTP) (here in the six-component membrane), which was identical for either one- or two-fold geranylgeranylated HVR.
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