Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.
Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.
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囊性纤维化跨膜电导调节器（CFTR）通道和多药耐药蛋白（MRP）泵跨膜域中的保守变构热点。
DOI：
10.1074/jbc.m114.562116
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Kirk,KevinL
中科院分区：
文献类型：
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作者：
Wei,Shipeng;Roessler,BryanC;Chauvet,Sylvain;Guo,Jingyu;Hartman4th,JohnL;Kirk,KevinL
三磷酸腺苷结合盒转运体(ABC)是一个古老的跨膜蛋白家族，它利用ATPase的活性来移动底物穿过细胞膜。ABC转运蛋白的ABCC亚家族包括活性药物出口蛋白(多药耐药蛋白(MRPs))和独特的ATP门控离子通道(囊性纤维化跨膜电导调节蛋白(CFTR))。CFTR通道与传统的配基门控离子通道具有相同的门控原理，但将其核苷酸结合域(NBD)上的ATP结合与其跨膜螺旋(TM)的构象变化相耦合的变构网络定义不够明确。目前也不清楚控制CFTR门控的机制是否与热力学上不同的MRP保守。在这里，我们报告了一类新的功能增益(GOF)突变，该突变位于毛孔衬里TM6的底部，是一个保守的脯氨酸。该氨基酸的多次取代促进了cftr的活性，并被弱激动剂5‘-腺苷-β，γ-亚胺二磷酸(AMP-PNP)激活。当TM6脯氨酸突变与先前报道的GOF突变相结合时，表现出相加的GOF效应，GOF突变位于TMS的外环，围绕着TMS的毛孔衬里。当引入具有缺陷的ATP结合的NBD突变体时，每个TM替换都变构地挽救了CFTR门控的ATP敏感性。这两类GOF突变也挽救了NBD中存在ATP结合缺陷的酵母MRP(York 1p)的缺陷药物出口。我们的结论是，保守的TM6脯氨酸有助于为CFTR通道开放和MRP介导的药物外流设置能量屏障，CFTR通道和MRP泵利用相似的变构机制将其转位途径的构象变化耦合到其NBD上的ATP结合。
ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.