Fig. 6: Peptide sequences compete with hRpn2 for hRpn13 but bind simultaneously with ubiquitin to allow bidentate interactions with ubiquitinated substrates.
From: An adaptive peptide-binding site in ubiquitin receptor hRpn13 revealed by structural studies
a Plot of the signal shifting for hRpn13_ΔMTT following the addition of 20-fold molar excess ENT or EDGEGEE. Dotted lines indicate one standard deviation above average and signals from the helix, terminal ends, or proteasome-binding site are colored blue, orange, and purple, respectively. b Expanded regions of 1H-15N HSQC spectra acquired on 0.1 mM 15N-labeled hRpn13_ΔMTT alone (black) or with equimolar unlabeled hRpn2 (green), 20-fold molar excess EDGEGEE (purple), or equimolar hRpn2 and 20-fold molar excess EDGEGEE (yellow). All spectra in this panel and (d) and (e) were acquired on a 600 MHz spectrometer equipped with a cryogenically cooled probe and at 25 °C. c Fluorescence polarization recorded during titration of unlabeled ENT (orange), EDGEGEE (purple), hRpn2 (940–953, light green) or hRpn2 (944–953, dark green) into 10 nM of TAMRA-labeled hRpn2 (940–953) in the presence of 80 nM hRpn13. Points are reported as mean ± SD, N = 6 (assay replicates). d Expanded regions of overlayed 1H-15N HSQC spectra acquired on samples of 0.1 mM 15N-labeled hRpn13_ΔMTT alone (black) or with equimolar ubiquitin (green), 20-fold molar excess EDGEGEE (purple), or both (blue). e Expanded regions of overlayed 1H-15N HSQC spectra acquired on samples of 0.1 mM 15N-labeled hRpn13_ΔMTT alone (black) or at 4-fold (left), 2-fold (middle) or equimolar (right) concentration with K48-diUbEDGEGEE (blue), K48-diUbENT (maroon) or K48-diUbD77 (green). f Cartoon of hRpn13 Pru (purple) interaction with a substrate (blue) ubiquitin chain (yellow) promoting its interaction with a disordered peptide sequence in the substrate. Source data for (a and c) are provided in the Source Data file.
