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.

中文翻译：

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序列和结构变异决定 WNK 激酶向 KLHL3 E3 连接酶的募集


BTB-Kelch 蛋白 KLHL3 是一种 Cullin3 依赖性 E3 连接酶，可介导激酶 WNK1-4 的泛素依赖性降解，从而控制血压和细胞体积。 KLHL3 的晶体结构定义了其与酸性降解决定子基序的结合，该基序包含在 WNK1、WNK2 和 WNK4 中严格保守的 PXXP 序列。第二个脯氨酸的突变消除了相互作用，导致 II 型假性醛固酮增多症高血压综合征。 WNK3 显示了一个包含四个氨基酸取代的分歧降解决定子基序，该基序去除了 PXXP 基序，引发了对其结合机制的疑问。为了理解这种非典型相互作用，我们确定了 KLHL3 Kelch 结构域与 WNK3 肽复合物的晶体结构。电子密度使得首次追踪完整的 11 聚体 WNK 家族降解决定子基序，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。总体而言，WNK3 肽采用了保守的结合姿势，只是为了适应结合界面处较大的氨基酸取代而进行了细微的转变。在中心，第二个脯氨酸被 WNK3 Thr541 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，其磷酸化会消除 KLHL3 相互作用，类似于引起高血压的突变。这些数据共同揭示了 KLHL3 Kelch 结构域如何适应多种 WNK 同工型的结合，并强调了 WNK3 招募的潜在调控机制。