Substrate-specific binding and conformational changes involving Ser313 and transmembrane domain 8 of the human reduced folate carrier, as determined by site-directed mutagenesis and protein cross-linking.
Substrate-specific binding and conformational changes involving Ser313 and transmembrane domain 8 of the human reduced folate carrier, as determined by site-directed mutagenesis and protein cross-linking.
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通过定点诱变和蛋白质交联测定涉及人还原叶酸载体的 Ser313 和跨膜结构域 8 的底物特异性结合和构象变化。
DOI：
10.1042/bj20100181
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Matherly,LarryH
中科院分区：
文献类型：
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作者：
Hou,Zhanjun;Wu,Jianmei;Ye,Jun;Cherian,Christina;Matherly,LarryH
RFC（还原性叶酸载体）是还原性叶酸和抗叶酸的主要转运体[例如MTX（甲氨蝶呤）]。RFC的特征是两半，每半都有6个由亲水性环连接的TMD（跨膜结构域）α螺旋，以及细胞质的N和c端。我们之前已经确定了tmd 4、5、7、8、10和11可以形成（抗）叶酸转运的亲水空腔。扫描半胱氨酸接近性方法显示，TMD8近端（位置311-314）与底物结合有关；ser313的半胱氨酸替代导致转运丧失。在本研究中，ser313突变为丙氨酸、半胱氨酸、苯丙氨酸和苏氨酸。突变型rfc在rfc缺失的R5 HeLa细胞中表达。用半胱氨酸或苯丙氨酸替代ser313可以消除MTX的转运，而在丙氨酸和苏氨酸突变体中保留了残余活性。在稳定的K562转染物中，与野生型RFC相比，S313A和S313T RFC的vmax值大幅降低，而MTX的inkt值没有变化。S313A和S313T rfc对十种不同（抗）叶酸底物的结合有不同的影响。通过在R5细胞中表达无半胱氨酸的TMD1-6 （N6）和TMD7-12 （C6）半分子，并在这些螺旋上插入半胱氨酸，然后用巯基反应性同源双功能交联剂处理，研究TMD8和TMD5之间的交联。所有半胱氨酸对均可见c6 - c6和n6 - n6交联。从n6和c6半胱氨酸对中，Cys175/ cys311是交联的；交联在转运底物的存在下增加。本研究的结果表明，TMD8的近端与TMD5并列，并且在转运底物存在时构象活跃，TMD8，包括Ser313，可能参与RFC底物结合域。
RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) α helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311–314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313resulted in loss of transport. In the present study, Ser313was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreasedVmaxvalues without changes inKtvalues for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1–6 (N6) and TMD7–12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6–C6and N6–N6cross-links were seen for all cysteine pairs. From the N6and C6cysteine pairs, Cys175/Cys311was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.