Interactome-Wide Prediction of Protein-Protein Binding Sites Reveals Effects of Protein Sequence Variation in Arabidopsis thaliana
A, C and E show the interacting partners of the proteins ZTL, CXIP1 and SHY2, respectively (interactions shown as dashed lines are not covered in the Arabidopsis Interactome data). B, D and F show a schematic representation of the sequences of the three proteins, including predicted binding sites (coloured box, using same colour as the proteins predicted to bind to it), mutagenesis sites (triangles for experimental mutagenesis sites, circles for naturally occurring sequence variants) and their positions, and residue surface accessibility (RSA) and conservation (bar plots) as predicted based on the sequence. A–B, in the protein ZTL, alanine mutagenesis of the residues 200 and 213 independently eliminate the interaction with ASK1; for ZTL, the stretch of residues from 208 to 220 is predicted as interaction site for binding with ASK2 and ASK4. This leads to the hypothesis that mutation on ZTP, specifically on the residue Leu213, would not only disrupt its interaction with ASK1, but also with other SKP1-like proteins, such as ASK2 and ASK4. C–D, In CXIP1, alanine mutagenesis of two highly conserved motifs (residues from 133 to 137; and residues from 97 to 100) leads to loss of ability to activate CAX1. For CXIP1, the stretch of residues from 125 to 136 was predicted as binding site, which overlaps the mutated motif SNWPT. The interaction of CXIP1 and the other interacting partners identified in the Arabidopsis interactome, i.e. AT5G09830, AT3G50780, AT1G70410 and TCP13 (AT3G02150), may also be mediated by the same motif. E–F, in the sequence of SHY2, three motifs were predicted as binding sites. The first (residues from 59 to 69; represented in grey) overlaps the position of two naturally occurring mutations (residues 67 and 69) and is predicted to be responsible for binding of TOPLESS (TPL, AT5G27030). A second motif (residues from 180 to 187; represented in brown) is predicted to be responsible for the interactions of SHY2 with six other IAA proteins. This leads to the hypothesis that two known mutations disrupt the interaction of SHY2 with TPL, but the same mutations do not impede its interaction with other IAA proteins.