DCAF8 binds DDB1 via an N-terminal helix-loop-helix motif to assemble CRL4 and promote cell cycle progression

Area of Science:

• * Molecular and Structural Biology
• * Cellular Biology
• * Biochemistry

Background:

• * Cullin-RING ligase 4 (CRL4) complexes are crucial E3 ubiquitin ligases that regulate numerous cellular processes.
• * Substrate specificity of CRL4 complexes is determined by DDB1-CUL4-associated factors (DCAFs).
• * The precise molecular mechanisms by which individual DCAFs interact with the adaptor protein DDB1 are not fully understood.

Purpose of the Study:

• * To determine the cryo-electron microscopy structure of DCAF8 in complex with DDB1.
• * To elucidate the molecular determinants governing CRL4^DCAF8 assembly.
• * To investigate the role of DCAF8-DDB1 interaction in cell cycle progression.

Main Methods:

• * Cryo-electron microscopy (cryo-EM) to determine the structure of the DCAF8-DDB1 complex at 2.53 Å resolution.
• * Biochemical assays to assess complex assembly and ubiquitination activity.
• * Cell-based assays to evaluate cell cycle progression defects.

Main Results:

• * The cryo-EM structure reveals that DCAF8 binds DDB1 via an N-terminal helix-loop-helix (HLH) motif.
• * This HLH motif inserts into a conserved pocket formed by the BPA and BPC domains of DDB1.
• * Disruption of the DCAF8 HLH-DDB1 interface impairs CRL4^DCAF8 assembly, reduces CDC25A ubiquitination, and causes cell cycle defects.

Conclusions:

• * A specific DCAF8 N-terminal HLH motif mediates recruitment to DDB1 within CRL4 ubiquitin ligase assemblies.
• * The identified DCAF8-DDB1 interaction interface is critical for CRL4^DCAF8 function and cell cycle regulation.
• * The double DxR box of DCAF8 is not involved in DDB1 binding and its role requires further investigation.