Unveiling the Distinct Mechanisms by which Disease-Causing Mutations in the Kelch Domain of KLHL3 Disrupt the Interaction with the Acidic Motif of WNK4 through Molecular Dynamics Simulation.
Unveiling the Distinct Mechanisms by which Disease-Causing Mutations in the Kelch Domain of KLHL3 Disrupt the Interaction with the Acidic Motif of WNK4 through Molecular Dynamics Simulation.
复制标题
通过分子动力学模拟揭示 KLHL3 Kelch 结构域中的致病突变破坏与 WNK4 酸性基序相互作用的独特机制。
DOI：
10.1021/acs.biochem.9b00066
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Peng,Ji-Bin
中科院分区：
文献类型：
--
作者：
Wang,Lingyun;Jiang,Chen;Cai,Ruiqi;Chen,Xing-Zhen;Peng,Ji-Bin
Kelch样3（KLHL 3）是E3泛素连接酶复合物的底物衔接子，其调节其底物的降解，包括无赖氨酸[K]激酶4（WNK 4）。KLHL 3的突变与II型假性醛固酮减少症（PHAII）有关，PHAII是一种遗传性高血压。许多导致PHAII的突变位于与WNK 4结合的KLHL 3的Kelch结构域中;然而，这些突变破坏结合的详细机制尚不清楚。在本研究中，我们使用分子动力学模拟和蛋白质印迹分析来研究这些突变对KLHL 3的Kelch结构域和WNK 4的酸性基序（AM）之间的相互作用的影响。除了L387 P突变导致KLHL 3对AM降解的失调，模拟结果与Western印迹分析结果相关良好，但模拟结果未重现。在模拟结果的基础上，Kelch结构域的结合表面上的突变通过两个主要机制影响Kelch-AM相互作用：改变AM结合位点的静电势和破坏Kelch-AM氢键。通过我们的模拟预测，Kelch结构域内部隐藏的突变对Kelch-AM相互作用没有影响或有适度的影响。埋藏突变R384 Q和S410 L破坏Kelch结构域内的分子内氢键，并间接影响Kelch-AM相互作用。未观察到埋藏突变A340 V或A494 T对AM降解或Kelch-AM相互作用的显著影响，这意味着这些突变可能破坏Kelch-AM相互作用以外的机制。
Kelch-like 3 (KLHL3) is a substrate adaptor of an E3 ubiquitin ligase complex that regulates the degradation of its substrates, including with-no-lysine [K] kinase 4 (WNK4). Mutations in KLHL3 are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. Many PHAII-causing mutations are located in the Kelch domain of KLHL3 that binds with WNK4; however, detailed mechanisms by which these mutations disrupt the binding are not well-understood. In the present study we use molecular dynamics simulations and Western blot analyses to examine the effects of these mutations on the interaction between the Kelch domain of KLHL3 and the acidic motif (AM) of WNK4. The simulation results correlated well with those from Western blot analyses with the exception of the L387P mutation, which led to deregulation of AM degradation by KLHL3 but not recapitulated by simulations. On the basis of the simulation results, a mutation on the binding surface of the Kelch domain affected the Kelch-AM interaction through two major mechanisms: altering the electrostatic potential of the AM binding site and disrupting the Kelch-AM hydrogen bonds. The mutations buried inside the Kelch domain were predicted by our simulations to have no or modest effects on the Kelch-AM interaction. Buried mutations R384Q and S410L disrupted intramolecular hydrogen bonds within the Kelch domain and affected the Kelch-AM interaction indirectly. No significant effect of buried mutation A340V or A494T on the AM degradation or Kelch-AM interaction was observed, implying these mutations may disrupt mechanisms other than Kelch-AM interaction.