Figure 3. UCH37–RPN13^C contacts the hydrophobic patches on both distal ubiquitin (Ub) units in a branched Ub₃ chain.

Residue-specific perturbations of backbone amide NMR signals in the (A) K48-linked distal Ub, (B) K6-linked distal Ub, (C) the proximal Ub, and (D) mutated proximal Ub(L8A,I44A) in branched K6/K48-linked Ub₃ caused by the addition of 1.2 molar equivalents of copurified UCH37(C88A)–RPN13^C. The NMR spectra are shown in Figure 3—figure supplement 1. Black bars represent chemical shift perturbations (CSPs, in ppm), gray bars mark residues exhibiting strong signal attenuations. Residues that were not observed or could not be unambiguously assigned/quantified due to signal overlap are marked with asterisks. Residues with strong signal attenuations or CSP >0.025 ppm are mapped (red) on the 3D structure of Ub (PDB code: 1UBQ); the hydrophobic patch residues are shown in sphere representation. (E) 1 µM UCH37–RPN13^C was incubated with 5 µM substrate as indicated at 37 °C. Reaction products were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie staining.

Figure 3—figure supplement 1. NMR spectra show unit-specific Ub₃ binding to UCH37–RPN13^C.

Overlays of ¹H–¹⁵N and ¹H–¹⁵N SOFAST-HMQC spectra (at 23 °C) of the individual ¹⁵N-labeled ubiquitin (Ub) units in the branched K6/K48-linked Ub₃ and of monomeric Ub before (blue) and after (green) the addition of a 1.2 molar equivalent of UCH37(C88S)–RPN13^C. Shown are fragments of the ‘fingerprint’ region of the spectra of (A) K48-linked distal Ub, (B) K6-linked distal Ub, (C) proximal Ub D77, and (D) proximal Ub D77 (L8A,I44A), as well as (E) monomeric WT Ub. Residues exhibiting significant signal attenuations as well as hydrophobic patch residues are indicated. The insets depict the ratio (I/I₀) of signal peak intensities after (I) and before (I₀) the addition of UCH37(C88S)–RPN13^C as a function of residue number. The spectra of unbound protein were recorded with 64 (A, B), 128 (C, D), or 32 scans (E), those of the complex were recorded with 2048 (A, B), 512 (C), and 256 (D, E) scans to compensate for the overall signal decrease as a result of binding. The overall attenuation of peak signal intensities (I/I₀) caused by the addition of UCH37(C88S)–RPN13^C was 0.09 ± 0.03 for K48-linked distal Ub and 0.14 ± 0.04 for K6-linked distal Ub (completely attenuated signals excluded), 0.43 ± 0.09 for the proximal Ub and 0.67 ± 0.07 for the proximal Ub with the L8A,I44A mutation (signals from flexible G76 and D77 excluded), and 0.73 ± 0.10 for monoUb. Residues that exhibited complete signal attenuation and those with I/I₀< mean − 2 × SD (in A, B) or I/I₀< mean − 1.5 × SD (in C) are marked with gray bars in Figure 3A–C.