Molecular determinants underlying substrate receptor specificity of human CRL4B E3 ubiquitin ligase

The vertebrate CRL4 family of Cullin-RING E3 ubiquitin ligases is distinguished from other cullin-based ligases by the presence of two highly homologous paralogs, CUL4A and CUL4B. Both CRL4 complexes use the DDB1 subunit to recruit dedicated and interchangeable substrate receptors called DCAFs, but the underlying mechanisms guiding DCAF specificity for CUL4B or CUL4A remain poorly understood. Here, we performed structural and biochemical analyses of the CRL4BLIS1 complex and identified two molecular determinants for CUL4B-specific DCAFs. First, we discovered that the unique CUL4B N-terminal extension directly binds CUL4B-specific DCAFs, enhancing their complex formation. This direct interaction can be modulated by phosphorylation, adding the possibility for spatiotemporal regulation. Second, the cryo-EM model of the CRL4BLIS1 complex identified a novel interface on the DDB1 subunit which promotes LIS1 recruitment. Quantitative affinity measurements and mutational analysis confirmed that this DDB1 interface is generally important for recruiting CUL4B-specific DCAFs including WDR1 and BRWD1, but not for CUL4A-specifc DCAFs, such as DCAF8. Together, our study identifies molecular determinants and unexpected interfaces on CRL4 components that dictate preference for DCAF recruitment.