Fig 1.
Position specific scoring matrix of LC8 binding motifs.
PSSM score of each position of the binding motif numbered from the central glutamine of the canonical “TQT” motif core. Amino acids corresponding to the ELM definition are framed. Values are color scaled as a heat map ranging from blue (negative) to red (positive) centered on zero (white). The Shannon entropy of each column is shown below in bitscore.
Fig 2.
Filtering protocol to find true binding partners.
(A) Schematic diagram of the binary filtering protocol we created utilizing information gain. A given attribute provides a binary split of the Parent group into the C1 and C2 child groups. The information gain (I) is then calculated as the difference of the Shannon entropy (H) of the Parent group minus the Shannon entropy of the Children groups weighted by their relative probabilities (p). These values were calculated over the dataset containing 40 known human binding partners and 10,000 random human segments from the proteome with a higher than zero PSSM score. (B) The information gain of the PSSM score (left panel) and four disorder prediction methods as a function of different cut-off values (right panel). The disorder prediction method used here were: IUPred (blue), Espritz Disprot (green) and VSL2 (red line), DISOPRED3 (cyan). Optimal cut-off values were obtained from the cut-off value corresponding to the maximum of the information gain, yielding 3.3 for the PSSM score, and 0.42 for IUPred disorder prediction score. (C) The outline of the final filtering protocol indicating the number of elements and percentage of cases in each Child group with the applied binary split.
Fig 3.
Summarized evolutionary conservation results of known human LC8 binding partners.
Protein names and motif start positions are indicated in the first and second columns. The colored boxes represent the presence of orthologues of the known partner at different evolutionary levels. The percentages and colour scheme of the boxes show the PSSM based motif conservation across all species. Conservation values increase from white (low motif conservation) to red (high motif conservation).
Fig 4.
Distribution of LC8 partners and predicted motifs.
(A) Distribution of LC8 interaction partners from PPI databases over the number of experiments. Partners that contain neither known nor predicted motifs are shaded grey according to the interaction type. (B) The composition of the high confidence prediction set shown as a Venn diagram. PPI: motifs in partners that appear in PPI databases. SLIM: motifs that show island-like conservation with the SLiMPrints method.
Fig 5.
GO enrichment in the high confidence set of LC8 binding partners.
Molecular Function (blue), Biological Process (green) and Cellular Component (red) categories that are enriched in the high-confident LC8 binding partners compared to human background. The x-axis represents the log-odds ratio of each enriched GO category. Process names related to the Hippo pathway are colored in red.
Table 1.
Experimentally validated LC8 binding motifs in the Hippo pathway.
Fig 6.
SPR analysis of the LC8 –AMOTL2 interaction.
(A) AMOTL2 peptide was injected to His6-LC8 immobilized on Tris-NTA sensor chip as described in Materials and Methods. Sensorgrams derived from five different analyte injections are indicated with colored lines. Black lines show the global fit of the experimental data to a 1: 1 Langmuir model. (B) Equilibrium analysis of the LC8 –AMOTL2 interaction at different LC8 immobilization levels.
Fig 7.
Structural organization and phylogenetic tree of the WWC family members.
The tree branch lengths do not represent real values. The subtrees of the three vertebrate WWC paralogs are highlighted by colored rectangles. Red and yellow boxes indicate the domains and coiled coil regions, respectively. Small purple and dark green boxes mark the location of the LC8 and PDZ binding motifs, respectively.
Fig 8.
Structural organization and phylogenetic tree of the AMOT family members.
The tree branch lengths do not represent real values. The subtrees of the three vertebrate AMOT paralogs are highlighted by colored rectangles. The angiomotin PFAM family was merged with overlapping coiled coil regions and indicated as angiomotin/Coil domain in orange. Other coiled coil regions and domains are colored yellow and red, respectively. Small boxes mark the location of linear motifs: brown (dark, light, medium)—WW binding, purple—LC8 binding, and dark green—PDZ binding motifs.