Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12
Chen Z., Wasney G., Picaud S., Filippakopoulos P., Vedadi M., D’Angiolella V., Bullock A.
ABSTRACT Wnt signaling is critically dependent on dishevelled proteins (DVL1-3), which are required to assemble an intracellular Wnt signalosome at the plasma membrane. The levels of dishevelled proteins are strictly regulated by multiple E3 ubiquitin ligases that target DVL1-3 for ubiquitination and proteasomal degradation. The BTB-Kelch family protein KLHL12 is a substrate-specific adaptor for a Cullin-RING E3 ligase complex that contains Cullin3 and Rbx1. KLHL12 was the first E3 ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP’ motif that is conserved in other known partners and substrates of KLHL12, including PLEXHA4, PEF1 and SEC31. A 20-mer peptide containing the DVL1 motif bound to the Kelch domain of KLHL12 with low micromolar affinity. In cells, the mutation or deletion of this motif caused a striking reduction in the binding, ubiquitination and stability of DVL1 confirming this sequence as a degron motif for KLHL12 recruitment. To determine the binding mechanism we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide. The structure revealed that the DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. Overall, these results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.