Fig. 7: Ub4a binding to the endo (UbB) and proximal Ub units of tetra-Ub chain with entire C-terminal tail deletion (ΔLRGG). | Nature Communications

Fig. 7: Ub4a binding to the endo (UbB) and proximal Ub units of tetra-Ub chain with entire C-terminal tail deletion (ΔLRGG).

From: Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation

Fig. 7

a Overlay of 1H-15N SOFAST-HMQC NMR spectra of the endo/next-to-proximal Ub (UbB) in [Ub]2-15NUbB-UbR72 free in solution (blue) and upon addition of Ub4a (red) in 1.5:1 molar ratio (peptide:polyUb). b Overlay of 1H-15N SOFAST-HMQC NMR spectra of the proximal Ub (UbR72) in [Ub]3-15NUbR72 free in solution (blue) and upon addition of Ub4a (red) in 2:1 molar ratio (peptide:polyUb). Spectral regions containing signals of the hydrophobic patch residues (L8, I44, V70) and the K48 isopeptide-bond signal (K48Z) are marked using orange and violet rectangles, respectively. Zoom in on these regions are shown below the respective full spectra. Signals of select residues are indicated, and their shifts are highlighted using straight lines. Insets on the bottom of the spectrum in a depict NMR signals of I13 and L67 in UbB at [Ub]2-15NUbB-UbR72:Ub4a molar ratios of 1:0 (blue), 1:0.5 (magenta), and 1:1 (red), to illustrate the slow-exchange regime of binding. c Residue-specific chemical shift perturbations (CSPs, Δδ) in the UbB unit of [Ub]2-15NUbB-UbR72 at the endpoint of titration with Ub4a (from a). Signals of C-terminal residues marked with light gray bars disappeared entirely. d Residue-specific CSPs in the proximal Ub (UbR72) of [Ub]3-15NUbR72 at the endpoint of titration with Ub4a (from b). The CSP of the K48 side-chain NH group involved in the isopeptide bond is shown at residue position 79. Note the different vertical scales of the plots in c and d. e Overlay of 1H-15N SOFAST-HMQC NMR spectra of the proximal Ub (UbA) in tri-Ub (blue) and of UbB in [Ub]2-15NUbB-UbR72 (red) at the endpoint of titration with Ub4a. Note the similarities between the blue and red spectra. The cartoon drawings to the right of e illustrate the peptide binding arrangements deduced from these NMR studies and highlight the interactions of Ub4a with the C-terminal tails and the isopeptide bonds of the indicated Ub units revealed by these data. Source data are provided as a Source Data file.

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