Domain mapping of the polycystin-2 C-terminal tail using de novo molecular modeling and biophysical analysis
Domain mapping of the polycystin-2 C-terminal tail using de novo molecular modeling and biophysical analysis
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DOI：
10.1074/jbc.m802743200
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发表时间：
2008-10-17
影响因子：
4.8
通讯作者：
Boggon, Titus J.
中科院分区：
文献类型：
--
作者：
Celic, Andjelka;Petri, Edward T.;Boggon, Titus J.
在多囊肾病（PKD）中，多囊蛋白-2（PC 2）在C-末端胞质尾（PC 2-C）中经常发生突变或截短。目前公认的PC 2-C模型由EF-手形基序与短卷曲螺旋重叠组成;然而，该模型未能解释PC 2截短该区域C-末端导致PKD的机制。此外，PC 2与肌醇1，4，5-三磷酸受体、KIF 3A和TRPC 1的直接结合需要PC 2-C中该区域以外的残基。为了解决这些差异并研究PC 2-C在PC 2功能中的作用，我们进行了从头分子建模和生物物理分析。使用ROBETTA服务器对PC 2-C进行从头分子建模，预测了如下两个结构域：EF-手基序（PC 2-EF）通过接头连接到先前未识别的C-末端卷曲螺旋（PC 2-CC）。该模型与当前模型有很大不同，并且与有限的蛋白水解、基质辅助激光解吸/电离质谱、N-末端测序和改进的卷曲螺旋预测算法相关。PC 2-C被拉长并通过PC 2-CC寡聚化，如通过分析性超离心和尺寸排阻色谱法所测量的，而PC 2-EF是球状和单体的。我们表明，PC 2-C和PC 2-EF具有微摩尔亲和力的钙（Ca 2+）的等温滴定量热法和进行Ca 2+诱导的构象变化的圆二色性。预测的EF-手环残基在PC 2突变为丙氨酸废除Ca 2+结合。我们的研究结果表明，PC 2-CC参与PC 2寡聚化，和PC 2-EF是一个Ca 2+敏感开关。PKD相关的PC 2突变位于可能破坏这些功能的区域，为PC 2突变如何导致疾病提供了结构性见解。
In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca2+) by isothermal titration calorimetry and undergo Ca2+-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca2+ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca2+-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease.