Structures of PROteolysis TArgeting Chimera (PROTAC)-mediated degradation machinery complex.

(A) Cereblon (CRBN) E3 ligase consists of an N-terminus domain (mint), helical binding domain (green), and C-terminus domain (gray). Bromodomain-containing protein 4 (BRD4BD1; targeted protein) consists of several important Lys residues (K72/76/99/102/111 black sticks) for successful degradation. (B) Degradation machinery complex is constructed with DDB1, CUL4A, NEDD8, Rbx1, E2, ubiquitin (Ub), and CRBN-PROTAC-BRD4BD1. (C) An illustration of the theoretical model of ubiquitination reaction. The catalytic site with multiple Asp residues creates a negatively charged environment that attracts Lys residues to enter the catalytic site. Lys and Asp residues then react with the C-terminus Gly of Ub for future degradation. (D) Chemical structure of each dBET PROTAC. aDegradation profile DC50/5h for four PROTACs was obtained from EGFP/mCherry reporter assay published in reference #5. bData published in reference #31. cData published in reference #5.

Flexibility of dBET PROTACs.

The last molecular dynamics (MD) frame from three MD runs was aligned with initial frame of the C-terminus domain (gray) of CRBN. Two warheads bound tightly to the respective proteins, whereas the linker is highly flexible, adopting various conformations. MD run 1 (red), MD run 2 (pink), MD run 3 (orange), and initial frame (black). (A) CRBN-dBET1_#35-BRD4BD1. (B) CRBN-dBET23_#14-BRD4BD1. (C) CRBN- dBET57_#9-BRD4BD1. (D) CRBN-dBET70_#91-BRD4BD1. (E) CRBN-dBET23xray-BRD4BD.

Number of ternary complexes used in the molecular docking and construction of degradation machinery complex.

List of molecular dynamics (MD) simulations for ternary complex CRBN-dBETx-BRD4BD1 and the degradation machinery complex. A1 and B2 indicate that the complex was assembled using scaffold cluster A1 and B1, respectively. The subscript number after each dBET degrader indicates the conformation index number from the protein–protein docking results. B1_dBET1_md3 indicates that a ternary conformation obtained from a CRBN-dBET1_#35- BRD4BD1 MD run (Figure 2 color orange) was used to build the initial conformation.

Functional dynamics and local interaction of each degradation machinery.

(A) K111 and D2132 of dBET1_#35 (Distance of K72/76 is reported in Figure S17). The left y-axis in blue presents the distance between Lys (N atom) of BRD4BD1 and Gly (C atom) of Ub, and the right y-axis in red presents the distance between Asp (O atom) of E2 and Gly (O atom) of Ub. The same left and right y-axis representations are used in C, E and G. (B) K72/76/111 of BRD4BD1 reaches the negatively charged catalytic site of E2. (C) K99 and D2133 of dBET23_#14. (D) K99 of BRD4BD1 reaches the negatively charged catalytic site of E2. (E) K102 and D2133 of dBET57_#9_MD1. (F) K102 of BRD4BD1 reaches the negatively charged catalytic site of E2. (G) K99 and D2103 of dBET70_#91. (H) K99 of BRD4BD1 reaches the negatively charged catalytic site of E2.

Quantifying the motion of degradation complex.

(A) Defining the pseudo dihedral angles, Linker(N3)-Leu975(Ca)-Gly1056(Ca)-Thr532(Ca) to capture the essential motion of the degradation complex. (B) Dihedral angle histogram shows the population distribution of the first 100 ns (blue) and last 100 ns (orange) of the MD simulation. The distribution shows a ∼10-15° dihedral angle shift.

Quantification of the correlated motion between the linker and degradation complex.

(A) Dihedral angles correlated with the degradation complex. r is the dihedral correlation coefficient (See Methods for detailed calculation). (B) Motion of BRD4BD1 and Ub indicates a shift between the initial frames (BRD4BD1, blue; Ub, yellow) and final frames (BRD4BD1, pink; Ub, orange) of MD simulation. K72, K99 and K102 engaged in the interaction with G75 of Ub in each degradation complex, which implies their potential for degrading BRD4BD1. For visualization purposes we present only K72 of dBET1_#35, but K76 and K111 can also engage in interaction with G75 (See Figure 3).