Nucleotide-induced conformational changes in molecular interaction fields.

<p><b>A:</b> 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>B:</b> The Hodgkin similarity index for the electrostatic potential <b>(top)</b> and the hydrophobic interaction field <b>(bottom)</b> 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.</p

All pairwise comparison of electrostatic potentials in human Rab GTPases.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034870#pone-0034870-g002" target="_blank">Figure 2B</a>, right.</p

Proposed sequential action of the guanine nucleotide exchange factor (GEF) on geranyl-geranylated Rab-GTPases.

<p>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.</p

Differences in the electrostatic and hydrophobic interaction fields of Rab7a and 7b.

<p><b>A</b>: 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>B</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.</p

Conservation of sequence and molecular interaction fields in human Rab GTPases.

<p><b>A</b>: 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>B:</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.</p

Rab GTPases as molecular switches.

<p><b>A</b>: 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>B</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 <i>H. sapiens</i> 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.</p

Clustering of human Rab proteins according to sequence and to electrostatic potential similarity.

<p><b>A (left)</b>: 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034870#pone.0034870.s001" target="_blank">File S1</a>. <b>B (right)</b>: Epogram. The proteins are clustered according to their distance in electrostatic potential space, , where SI_ab 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.</p

Pipeline and schematic illustration of the integrative analysis.

<p>(<b>a</b>) Overview of the analysis. (<b>b</b>) Schematic of the two integrative methods. They combine phenotypic information from RNAi screening data (heatmaps) with either known protein complexes (top) or binary interaction networks (bottom). Each gene (A through G) has been assayed with several different siRNAs, i.e., several profiles are obtained. The heatmap thus shows the different profiles per gene using a color code (deviation from average); each column represents a phenotype parameter (such as number or size of endosomes). In the case of sets (top) our method assesses all profiles associated with all genes in the set and identifies an over-represented profile. In the case of network data (bottom) the method searches for network modules (sub-networks) enriched for a common profile. In both cases the algorithm determines a ‘references’ profile representing the common profile of the set or network module. Note that in the network case we also consider anti-correlated profiles, as genes might have opposing effects on the readout. For the set-based analysis, significance is determined through appropriate randomizations that take into account the number of genes and profiles in each complex (<b>c</b>). For the network-based analysis, the significance of each module is estimated by a semi-analytical approach that takes into account the composition of the module at any expansion step and the probability of observing specific patterns in the dataset (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003801#s1" target="_blank">Methods</a> for details).</p

Selected network modules with enriched phenotypes.

<p>See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003801#pcbi-1003801-g004" target="_blank">Figure 4</a> legend for more details. The topology of the extracted sub-networks is shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003801#pcbi.1003801.s014" target="_blank">Figure S14</a>. Note that genes in the same module (as opposed to protein complexes) can have anti-correlated profiles, but selected profiles of the same gene must be positively correlated. The profiles shown represent the direction taken by the majority of the genes in the module. E.g., the phenotype of EGFR is anti-correlated to the reference profile shown for the EGFR-IGFR module.</p

Selected protein complexes with enriched phenotypes.

<p>Each complex is represented by its reference profile, which is computed as the most consistent signature across all members of the complex. The analysis is based on 19 parameters for each channel (EGF and transferrin, TF) and two co-localization parameters (TF with EGF and EGF with TF). The x-axis shows the difference between the Euclidean (or L2) norm of the transferrin and the EGF parameters the y-axis represents the strength of the phenotype computed as the sum of the norm of both the transferrin and the EGF signals . Each protein complex is shown as a circle, its color reflecting the primary direction of the phenotype (value on x-axis). Inset bar-plots show summary versions of the reference phenotypic profiles by projecting them from 40 to 14 dimensions (red: response on EGF channel, green: response on transferrin channel). See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003801#pcbi.1003801.s020" target="_blank">Table S1</a> for details on the parameters. More details about complex membership are shown in Supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003801#pcbi.1003801.s013" target="_blank">Figure S13</a>.</p