Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin 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 PLEKHA4, PEF1, SEC31 and DRD4. 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 that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. 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.

中文翻译：

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PGXPP degron基序的鉴定在其与E3连接酶KLHL12结合的不完整和结构基础上。

Wnt信号转导依赖于散乱的蛋白（DVL1-3），该蛋白在质膜上组装细胞内Wnt信号小体。DVL1-3的水平受多种介导其泛素化和降解的Cullin-RING E3连接酶的调节。BTB-Kelch蛋白KLHL12是第一个被鉴定用于DVL1-3的E3泛素连接酶，但是决定其底物相互作用的分子机制仍然未知。在这里，我们将DVL1-3的相互作用映射到“ PGXPP”基序，该基序在KLHL12的其他已知伙伴和底物（包括PLEKHA4，PEF1，SEC31和DRD4）中保守。为了确定结合机理，我们解决了与低微摩尔亲和力结合的DVL1肽复合的KLHL12的Kelch域的2.4晶体结构。DVL1底物采用了U形转弯构型，该构型使得能够与Kelch域β-螺旋桨的所有六个叶片发生疏水性相互作用。在细胞中，该基序的突变或缺失减少了DVL1的结合和泛素化，并增加了其稳定性，从而确认了该序列为KLHL12募集的德格隆基序。这些结果定义了确定由KLHL12调节DVL的分子机制，并建立了KLHL12 Kelch域，将其作为富含脯氨酸的新基序的新蛋白质相互作用模块。