Figure 4

Computational modelling and mutational validation of the Galectin-1/RBD complex.

(A) Computational representation of a monomer of Gal-1 (3W58) and C-Raf-RBD (1C1Y: herein RBD)⁵² complex from an optimized low energy molecular docking pose superimposed with dimeric Gal-1 from the same PDB deposition. Numbering of residues is according to sequences deposited in UniProt (P09382 – Gal-1_Homo sapiens, P04049 – C-Raf_Homo sapiens). The loop (loop 4) that undergoes major conformational and stereo-chemical changes between apo- and liganded Gal-1 is coloured orange (Supplementary Fig. 3A). Left: Note that the Gal-1 dimer interface, marked by the four mutated residues, is to the left near the N-terminus (N) of Gal-1. Residues forming the CBS are shown on the left monomer. The grey oval marks the region on the C-Raf-RBD that contacts Ras. Enlarged panel to the right shows a close-up view into the putative protein-protein interface and major interactions. Residues that were mutated in the RBD and showed an effect are marked with asterisks. The uncertainty regarding the interacting surface on Gal-1 is indicated by the translucent grey box. (B) Representative confocal images of HEK293-EBNA cells co-transfected with mGFP-Gal-1 and mRFP-C-Raf-RBD (RBD) mutated in the indicated residues. Columns represent imaged fluorescent channels, appropriate for the indicated construct. The nucleus is stained by DAPI. Overlay images show superposition of images to the left. Scale bar is 5 μm. (C) Interaction between mCit-Gal-1 (left) or mGFP-Gal-1 (right) and mRFP-tagged C-Raf-RBD and derived interfacial mutants studied using FLIM-FRET in HEK293-EBNA cells transiently expressing indicated constructs (three independent biological experiments). (D) Interaction between mGFP-H-rasG12V and mRFP-tagged C-Raf-RBD and derived interfacial mutants with or without coexpressed non-labelled Gal-1 (+Gal-1) studied using FLIM-FRET in HEK293-EBNA cells transiently expressing indicated constructs (three independent biological experiments). (C,D) Plotted values correspond to the mean ± SEM. Numbers inside the bars indicate total number of cells imaged. The Methods section describes indicated statistical comparisons (ns, non significant; *p < 0.05; **p < 0.01; ***p < 0.001). Samples with coexpressed fluorescent proteins mCit and mRFP (C) or mGFP and mRFP (C,D) served as a FRET control. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample.