Unraveling the Engagement of Kinases to CRBN Through a Shared Structural Motif to Optimize PROTACs Efficacy

Abstract

PROteolysis TArgeting Chimeras (PROTACs) offer a therapeutic modality for protein target engagement, exploiting the ubiquitin-proteasome system to achieve precise degradation of a protein of interest. Recent advancements in understanding the structural biology of the CRL4A E3 ligase complex, particularly its recruitment of neo-substrates through the G-loop motif, have provided valuable insights into the optimization of PROTAC efficacy. This perspective delves into the molecular determinants governing PROTAC selectivity and degradation efficiency, with a specific focus on kinases showing distinct G-loop conformations. By employing computational approaches to predict ternary complexes, along with the identification of binding patterns, it is possible to address limitations posed by structural data scarcity, thereby enhancing rational design strategies.

Keywords: E3 ligases; G-loop; PROTACs; kinases.

Figure 1

(a) Schematic representation of the ubiquitination process involving the ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner, through the formation of a thioester bond. The activated ubiquitin is transferred to the ubiquitin-conjugating enzyme (E2), which delivers ubiquitin to the ubiquitin ligase (E3) for substrate selection. E3 simultaneously binds the E2–ubiquitin complex and the substrate protein, facilitating the covalent attachment of ubiquitin to a lysine residue on the target protein, thereby marking it for proteasomal degradation. PROTACs (proteolysis-targeting chimeras) facilitate targeted degradation by serving as bifunctional molecules that recruit E3 ligases and bring them into proximity with the protein of interest (POI), promoting its ubiquitination and subsequent degradation. (b) Schematic and structural representation of the CRL4A^CRBN complex. DDB1 (A, B, C): DNA Damage-Binding Protein 1 subunits A, B, and C; CRBN: cereblon; POI: protein of interest; UBE2D: ubiquitin-conjugating enzyme E2; RBX1: RING protein; NEDD8: ubiquitin-like protein; UBC: polyubiquitin-C. Rotating arrows around DDB1 subunits A and C and around CUL4A represent the rotational movement of these subunits relative to subunit B and the opening motion involving CUL4A, respectively. In the rows below, the three stable conformations of the complex, i.e., linear, twisted, and hinged, are reported. In the linear orientation, used as a reference, the broad face of BPB aligns similarly to BPC, whilst the hinged and twisted are rotated ~70° and ~140° from the linear one, respectively. The entire complex (top row). DDB1-A-C subunits (bottom row) highlight the rotational differences between subunits A and C. Color legend: CRBN (light pink), CUL4A (light blue), UBE2D (purple), UBC (light yellow-green), RBX1 (white), and NEDD8 (dark green) are shown in the surface representation, while DDB1-A-B-C subunits (light green) are in the cartoon representation.

Figure 2

(a) Schematic representation of the G-loop (contacts are shown as dashed yellow lines), which consists of a β-hairpin loop with a glycine residue in a key position (G0), and (b) the superimposed G-loops from experimentally solved structures highlighting the interaction with CRBN.

Figure 3

(a) Models of the CDK kinases’ interaction with the CRL4A^CRBN complex in the hinged conformation; “CDK” label represents both CDK4 and CDK6. (b) Model of the productive ternary complex when PROTAC-6 is docked into the cleft (coloring scheme is consistent with panel). (c) Comparison of the PROTAC-mediated binding mode (contacts shown in dashed yellow lines) between (c) the CDK6 G-loop (32-DLKNG-36, orange) and (d) the CDK4 G-loop (25-DPHSG-29, beige), which are predicted to interact with CRBN (light pink) in a cleft formed by residues Asn351, Tyr355, His357, and Trp400.