Fig. 3: DARPin binding epitopes and GS-eGFP ubiquitination.
From: Molecular features defining the efficiency of bioPROTACs
a Mass spectrometric analysis (MS-MS) of ubiquitination sites after in vitro ubiquitination of GS-eGFP, induced by DP6-based bioPROTAC. The sample was digested in the three indicated ways to improve coverage. Ubiquitinated lysines carry a covalent GG or LRGG adduct and are circled in different colors. For representative spectra, see Supplementary Fig. S27. b Structure of eGFP (gray, PDB ID: 5MAD) in complex with DP6 (yellow, PDB ID: 5MA6) and DP9 (blue, PDB ID: 5MAD) created in PyMOL. To create bioPROTACs, the CHIPΔTPR domain is genetically fused at the indicated C-terminus. GS-eGFP N- and C-termini are shown in black and purple, respectively. eGFP lysine ubiquitination sites upon DP6-bioPROTAC engagement are indicated in the same colors as shown in (a). The C-terminal K241 is not indicated, since the residue is not resolved in the structure, but close to the indicated C-terminus (M233, purple). c–e Assays involving GS-eGFP mutants. GS-eGFP_mut1: K104R_K159R, GS-eGFP_mut2: K6R_K104R_K159R, GS-eGFP_mut3: K104R_K159R_K241R, GS-eGFP_mut4: K6R_K104R_K159R_K241R. c Western-blot of InVitroUb ubiquitination assay of DP6-bioPROTAC in complex with indicated GS-eGFP lysine mutants. Samples were detected with either anti-eGFP antibody or anti-DARPin polyclonal antibody to show GS-eGFP or bioPROTAC ubiquitination. To image marker bands and antibody-stained bands, the same membranes were imaged in two separate channels. Uncropped membranes are shown in Supplementary Fig. S17. d Degradation rate constants determined by microinjection into HEK293 cells of GS-eGFP and GS-eGFP mutants alone (/) or in complex with DP6-bioPROTAC or inactive DP6-bioPROTACR272A mutant. Shown are average degradation rate constants of single cells. Error bars represent standard deviations. Numbers of analyzed cells per analyte are shown in Supplementary Table ST1. The four lysines or arginines within the GS-eGFP mutants are color-coded according to mutated lysine residues as shown in (a) and (b). e Western-blot of LysateUb ubiquitination assay of DP6-bioPROTAC or inactive DP6-bioPROTACR272A mutant alone or in complex with indicated GS-eGFP lysine mutants. Samples were detected by anti-eGFP antibody to show GS-eGFP ubiquitination. f–k Structures of indicated DARPins (blue) in complex with eGFP (gray) determined by x-ray crystallography. PDB IDs: DP1 9F22, DP2 9F23, DP3 6MWQ, DP4 9F24, DP6 5MA6 (differs in 5 mutations in the C-cap compared to our construct), DP9 5MAD. Models of DP5, DP7, and DP8 are shown in Supplementary Fig. S30. Crystallographic data are shown in Table 2. For d, the number of analyzed cells are shown in Supplementary Table ST1.
