Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12
Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12
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DOI：
10.1098/rsob.200041
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发表时间：
2020-06-24
期刊：
影响因子：
5.8
通讯作者：
Bullock, Alex N.
中科院分区：
文献类型：
--
作者：
Chen, Zhuoyao;Wasney, Gregory A.;Bullock, Alex N.
Wnt信号传导依赖于杂乱蛋白（DVL 1 -3），其在质膜上组装细胞内Wnt信号体。DVL 1 -3的水平受多个Cullin-RING E3连接酶调节，这些连接酶介导DVL 1 - 3的泛素化和降解。BTB-Kelch蛋白KLHL 12是第一个被鉴定为DVL 1 -3的E3泛素连接酶，但决定其底物相互作用的分子机制仍然未知。在这里，我们将DVL 1 -3的相互作用定位到一个“PGXPP”基序，该基序在KLHL 12的其他已知伴侣和底物中是保守的，包括PLEKHA 4，PEF 1，SEC 31和DRD 4。为了确定结合机制，我们解析了KLHL 12的Kelch结构域与DVL 1肽复合的2.4埃晶体结构，所述DVL 1肽以低微摩尔亲和力结合。DVL 1底物采用U形转弯构象，其能够与Kelch结构域β-螺旋桨的所有六个叶片发生疏水相互作用。在细胞中，该基序的突变或缺失降低了DVL 1的结合和泛素化，并增加了其稳定性，证实了该序列作为KLHL 12募集的降解决定子基序。这些结果定义了决定DVL调节KLHL 12的分子机制，并建立了KLHL 12 Kelch结构域作为一个新的蛋白质相互作用模块的一个新的脯氨酸丰富的基序。
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 angstrom 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 beta -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.