Figure 1: NMR mapping the interaction with SUMO-2 chains on the RNF4–SIM region.

(a) Amino-acid sequence for the N-terminal fragment of RNF4 used in this study. The four SIMs as well as ‘SIM5’ are underlined; the residues affected by titration are coloured red, whereas those unaffected are coloured blue; residues coloured black are not classified owing to lack of assignment or overlap. A dashed line over the affected residue means its position was shifted with increasing SUMO concentrations; a thin line means the peak was initially shifted, then disappeared after ~1:1 SUMO added; a thick line indicates that the peak disappeared when <0.3 molar equivalents of SUMO was added. A schematic representation for RNF4 is shown below the sequence, where the SIMs are supposed to form β-strands upon binding to SUMO; a very positive region (with eight arginines) and a negative stretch (the two serine residues may be phosphorylated) are also highlighted in the scheme. (b) Overlay of ¹H–¹⁵N HSQC NMR spectra for free RNF4 32–133 (red), RNF4 32–133 titrated with SUMO monomer (magenta), SUMO dimer (cyan), SUMO dimer with truncations (yellow) and polySUMO chain (blue); the titrations are represented by the mixtures at 1 M equivalent SUMO (in monomer concentration). Labels are placed close to the peaks in the free state. The full spectra are also available as Supplementary Fig. 2.