Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase.
Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase.
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决定WNK激酶募集至KLHL3 E3连接酶的序列和结构变异。
DOI：
10.1042/bcj20220019
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发表时间：
2022-03-18
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BTB-Kelch蛋白KLHL 3是Cullin 3依赖性E3连接酶，其介导激酶WNK 1 -4的泛素依赖性降解以控制血压和细胞体积。KLHL 3的晶体结构已经定义了其与含有在WNK 1、WNK 2和WNK 4中严格保守的PXXP序列的酸性降解决定子基序的结合。第二个脯氨酸的突变消除了引起高血压综合征假性醛固酮减少症II型的相互作用。WNK 3显示出含有四个氨基酸取代的趋异的降解决定子基序，其去除了PXXP基序，从而引起了关于其结合机制的问题。为了理解这种非典型的相互作用，我们确定了与WNK 3肽复合的KLHL 3 Kelch结构域的晶体结构。电子密度使完整的11-mer WNK-家族降解决定子基序首次被追踪，揭示了先前工作中未捕获的几个保守特征，包括额外的盐桥和氢键相互作用。总的来说，WNK 3肽采用保守的结合姿势，除了在结合界面处适应体积较大的氨基酸取代的微妙变化。在中心，第二个脯氨酸被WNK 3 Thr 541取代，在WNK家族降解决定子中提供了独特的可磷酸化残基。荧光偏振和结构建模实验表明，其磷酸化将废除KLHL 3的相互作用类似于高血压引起的突变。总之，这些数据揭示了KLHL 3 Kelch结构域如何适应多种WNK亚型的结合，并突出了WNK 3募集的潜在调控机制。
The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1–4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing four amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.