Figure 5: NMR chemical shift mapping of RNF4-SIM2,3 interaction with lys11-linked diSUMO-2.
From: Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

(a) 1H–15N HSQC spectra of SUMO-2 dimer with the distal domain (SUMO-2 ΔN11,cyan) labelled and titrated with increasing molar equivalents of SIM2,3 peptide. Large chemical shift changes in response to increase amount of the SIM2,3 peptide indicate a specific interaction that is fast-intermediate exchange on the NMR timescale. (b) Plot of weighted chemical shift perturbations calculated using [(1H difference)2+((15N difference)1/5))2]0.5 versus residue number for the distal domain; those with changes >0.1 p.p.m. indicate likely proximity to the binding site. (c) Map of the significant chemical shift changes (>0.1 p.p.m.) are coloured red on a hybrid cartoon-surface representation of the distal domain structure. This highlights binding of the SIM2,3 peptide to the canonical SIM-binding site between second β2-strand and the α-helix. (d) Overlay of 1H–15N HSQC spectra for distal (cyan) and proximal (green) domains with the SUMO-2 dimer in complex with SIM2,3 shows that very similar chemical shift changes occur for both subunits except for residues close to the isopeptide bond (that is, N14 and D15 shown here). The high degree of similarity between chemical shifts of SIM-binding site residues for both distal and proximal SUMO-2 domains indicate that direction of the SIM2,3 peptide (N- to C terminus) relative to diSUMO-2 chain (distal to proximal) is exchangeable.