BTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer, and Alzheimer's disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML, and ULK1. We identified an "LPDLV"-containing motif in the DAPK1 death domain that determines its recruitment and degradation by KLHL20. A 1.1-Å crystal structure of a KLHL20 Kelch domain-DAPK1 peptide complex reveals DAPK1 binding as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the β propeller. Here, KLHL20 forms salt-bridge and hydrophobic interactions including tryptophan and cysteine residues ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of β-propeller domains versus linear grooves and suggests a new target for structure-based drug design.
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BTB, CUL3, Cullin-RING ligase, autophagy, cancer, crystallography, protein-protein interaction, ubiquitination, Adaptor Proteins, Signal Transducing, Binding Sites, Crystallography, X-Ray, Death-Associated Protein Kinases, Female, HEK293 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Protein Binding, Protein Domains, Protein Structure, Secondary, Proteolysis, Ubiquitination