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. 2012;7(4):e34870.
doi: 10.1371/journal.pone.0034870. Epub 2012 Apr 16.

The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity

Matthias Stein  1 , Manohar Pilli, Sabine Bernauer, Bianca H Habermann, Marino Zerial, Rebecca C Wade
Affiliations

The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity

Matthias Stein et al. PLoS One. 2012.

Abstract

Background: Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.

Methodology/principal findings: Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.

Conclusions/significance: We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Rab GTPases as molecular switches.
A: Schematic figure showing the interactions of a Rab-GTPase with its effector proteins. The Rab protein is geranyl-geranylated near the C-terminus to enable membrane binding. Guanine nucleotide exchange factor proteins (GEFs) accelerate the GDP to GTP exchange and thus convert the GTPase from its inactive into its active form. Guanine nucleotide activating proteins (GAPs) deactivate the Rab GTPase by facilitating the intrinsic GTP hydrolysis. B: Sequence and structural mapping of characteristic segments of Rab proteins. Rab family-specific (RabF1-RabF5) sequence segments that are distinct for Rab GTPases and distinguish them from other small GTPases of the Ras superfamily (Pereira-Leal and Seabra, 2001) are mapped onto the crystal structure of the Rab5A GTPase from H. sapiens in an active conformation (Terzyan et al., 2004) (displayed in orange-red). Rab subfamily (RabSF1-RabSF3) specific sequence segments are characteristic for subsets of the Rab GTPases in which each subfamily displays a high sequence identity (displayed in green) (Pereira-Leal and Seabra, 2000). The nucleotide is shown in pink. The Switch I and II regions, which undergo large nucleotide dependent conformational transitions, are labeled.
Figure 2
Figure 2. Clustering of human Rab proteins according to sequence and to electrostatic potential similarity.
A (left): Unrooted phylogenetic tree based on a Gblocked alignment of the sequences. The tree shows six subclusters. The color coding corresponds to the sequence-based phylogenetic analysis by Colicelli (Colicelli, 2004). For a phylogenetic tree derived from analysis of the full-length sequences, see File S1. B (right): Epogram. The proteins are clustered according to their distance in electrostatic potential space, formula image, where SIab is the pairwise Hodgkin similarity index (Hodgkin and Richards, 1987) calculated for the complete protein skin. The electrostatic potential distance clustering suggests six subclusters with a different composition from the sequence-based analysis.
Figure 3
Figure 3. All pairwise comparison of electrostatic potentials in human Rab GTPases.
Heat-map representation of the distance matrix of electrostatic potentials computed for the entire protein skins of human Rab proteins with the color code ranging from red (identical) to blue (dissimilar). The ordering of the Rab GTPases corresponds approximately to that of Figure 2B, right.
Figure 4
Figure 4. Differences in the electrostatic and hydrophobic interaction fields of Rab7a and 7b.
A: Electrostatic isopotential contours at ±0.5 kT/e (blue: positive, red: negative) around cartoon representations of Rabs 7a and 7b. Each protein is shown in two views, one focusing on the switch I and II regions and the other rotated 180° about the vertical axis. B: Conservation of electrostatic and hydrophobic interaction fields mapped onto the protein crystal structure as a color gradient from blue (variable) through green to red (conserved) on a scale from −1 to +1 for electrostatic potentials and 0 to +1 for hydrophobic fields. The nucleotide is shown in pink. For the regions in black, no hydrophobic similarity index was computed due to high polarity.
Figure 5
Figure 5. Conservation of sequence and molecular interaction fields in human Rab GTPases.
A: Rab5a (PDB entry 1R2Q) secondary structure and amino acid residues colored from variable (blue), through intermediate (green) to conserved (red) according to conservation of sequence (Seq), electrostatic potential (EP) and hydrophic interaction field (HIF). B: Cartoon representation of Rab5a with bound nucleotide in stick representation (pink) and the similarity of the electrostatic (EP) and hydrophobic interaction fields (HIF) mapped onto the structures. The conservation scores of the corresponding residues are represented by a color gradient from blue (variable), through green to red (conserved). Each structure is shown in two views, one focusing on switch regions I and II and one rotated 180° about the vertical axis to highlight the helices and CDRs.
Figure 6
Figure 6. Structural mapping of molecular interaction field conservation in human Rab GTPase subclusters.
Simplified Rab family tree in which representatives of subclusters from the electrostatic potential comparison (Fig. 2B) are displayed with the similarity of molecular interaction fields among the subcluster members. Cartoons are shown in two views, the ones close to the branch focus on the switch I and II regions and the ones further away are shown rotated by 180° around the vertical axis. The nucleotide is shown in a pink stick representation. The average of all pair-wise SI scores of all members of each subfamily is mapped onto the protein structure as a color gradient from blue (variable), through green (intermediate) to red (conserved) for A: electrostatic potential and B: hydrophobic interaction field. Positive HIF energies were neglected for similarity calculations and those regions are represented in black. For an alternative subclustering according to the sequence-based phylogentic tree, see also File S1.
Figure 7
Figure 7. Nucleotide-induced conformational changes in molecular interaction fields.
A: Differences in conformation and electrostatic potential between the inactive GDP-bound and active GTP-bound forms of human Rab5a. Isocontour plots at levels of +1 kT/e (blue) and −1 kT/e (red). The largest conformational change is associated with the switch II region (formation of a C-terminal alpha-helical region) and the switch I region (tight binding of GTP). B: The Hodgkin similarity index for the electrostatic potential (top) and the hydrophobic interaction field (bottom) within a radius of 15 Å around the Cα atoms of the inactive (GDP-bound) and active (GTP-bound) human Rab proteins is plotted against residue number.
Figure 8
Figure 8. Proposed sequential action of the guanine nucleotide exchange factor (GEF) on geranyl-geranylated Rab-GTPases.
The GEF destabilizes the ternary GDP-Rab-GEF complex and GDP is released. The nucleotide-free Rab and the GEF form a stable binary complex. Subsequent GTP binding to the nucleotide-free Rab again destabilizes the ternary complex, the GEF is released and the Rab is left in its active GTP-bound state.
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