Figure 4. CSPs in ¹⁵N-UbcH5a caused by either RNF125^stop99 or RNF125^stop129.

(A) Overlay of ¹H-¹⁵N HSQC spectra of 0.18 mM ¹⁵N-UbcH5a before (black), and after (red) addition of 0.5 molar equivalents of RNF125^stop99 (left) or RNF125^stop129 (right). Bar graphs represent the combined ¹H and ¹⁵N chemical shift perturbations of ¹⁵N-UbcH5a at these molar ratios. The red bars indicate peaks that have broadened beyond detection. Pro residues are indicated by (*). (B) Expanded regions of ¹H-¹⁵N HSQC spectra for 0.18 mM UbcH5a showing specific peaks before (black) and after the addition of RNF125^stop129 at 0.125 (blue), 0.25 (grey), 0.5 (pink), 0.9 (red) molar equivalents. Peaks for residues at known RING binding sites are shown: I6 in helix 1, K63 in loop 4, L97 in loop 7; C85 is the active site Cys; I88 undergoes allosteric changes upon RING binding; L103 is present in helix 2.(C) Combined ¹H and ¹⁵N chemical shift perturbations of 0.18 mM ¹⁵N-UbcH5a at 0.9 molar equivalents of RNF125^stop129. Assigned residues are in black in the sequence underneath the graph, Pro are indicated by a (*). Red bars indicate residues that broadened beyond detection in the presence of RNF125^stop129. The blue bars below the graph indicate areas that have been published to undergo extensive CSPs in similar NMR titrations: residues 1–12 in helix 1, 58–63 in loop 4 and 94–99 in loop 7. A schematic representation of secondary structure elements (α-helices as cylinders and β-sheets as arrows) of UbcH5a is also shown.