Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.
Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.
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二硫键和 N-糖基化之间的相互作用定义了 SLC4 Na 偶联转运蛋白的细胞外拓扑。
DOI：
10.1074/jbc.m114.619320
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Kurtz,Ira
中科院分区：
文献类型：
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作者：
Zhu,Quansheng;Kao,Liyo;Azimov,Rustam;Abuladze,Natalia;Newman,Debra;Kurtz,Ira
SLC4 Na+偶联转运体的细胞外环3 （EL-3）包含4个高度保守的半胱氨酸和多糖基化共识位点。在电致Na+-HCO3−共转运体NBCe1-A中，EL-3是最大的胞外环，预计由82个氨基酸组成。为了确定NBCe1-A EL-3中保守的半胱氨酸和n -糖基化位点的结构功能重要性，我们分析了EL-3二硫键和n -糖基化之间的潜在相互作用及其在EL-3拓扑折叠中的作用。我们的研究结果表明，这4个高度保守的半胱氨酸形成了两个分子内二硫键，分别是cys583 - cys585和Cys617-Cys642，它们以折叠构象约束EL-3。第二二硫键的形成是自发的，不受EL-3的糖基化状态或第一二硫键的影响，而第一二硫键的形成依赖于第二二硫键的存在，并受到n -糖基化的影响。重要的是，来自每个单体的EL-3邻接位于NBCe1-A二聚体界面。当两个二硫键缺失时，EL-3采用一种易于蛋白酶消化的延伸构象。来自每个单体的两个对称折叠EL-3环的独特相邻平行位置类似于一个域样结构，这对NBCe1-A在体内的功能可能很重要。此外，这种独特结构的形成严重依赖于膜处理NBCe1-A二聚体中二硫键和n -糖基化之间精细调节的相互作用。细胞外环3 （EL-3）中的半胱氨酸在SLC4 Na+偶联转运体中高度保守。结果二硫键和n -糖基化决定了NBCe1-A EL-3的独特形貌。结论EL-3在NBCe1-A二聚体界面上形成了类畴结构。意义二硫键和n -糖基化的相互作用可能在跨膜蛋白成熟过程中起重要作用。
The extracellular loop 3 (EL-3) of SLC4 Na+-coupled transporters contains 4 highly conserved cysteines and multipleN-glycosylation consensus sites. In the electrogenic Na+-HCO3−cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and theN-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding andN-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys583-Cys585and Cys617-Cys642, respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by theN-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected byN-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A functionin vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding andN-glycosylation in the membrane processed NBCe1-A dimer.BackgroundCysteines in extracellular loop 3 (EL-3) are highly conserved in SLC4 Na+-coupled transporters.ResultsDisulfide bonding andN-glycosylation uniquely determine the topography of NBCe1-A EL-3.ConclusionA domain-like structure formed by EL-3 is present at the NBCe1-A dimeric interface.SignificanceThe interplay between disulfide bonding andN-glycosylation may play an important role in transmembrane protein maturation.