Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers
Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers
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DOI：
10.1074/jbc.m112.357590
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发表时间：
2012-07-27
影响因子：
4.8
通讯作者：
von Heijne, Gunnar
中科院分区：
文献类型：
--
作者：
Lloris-Garcera, Pilar;Bianchi, Frans;von Heijne, Gunnar
细菌多药转运蛋白EmRE是一种双重拓扑膜蛋白，因此能够以两个相反的方向插入到膜中。EmRE的功能形式是同源二聚体；然而，二聚体中各亚基的相对定位仍在争论中。使用膜上具有固定相反取向的Emre变体，我们现在表明，尽管蛋白质能够形成平行的二聚体，但二聚体中的亚单位的反平行组织是首选的。对完整低聚物和二硫键交联的Blue-Native PAGE分析表明，在膜中，只有在没有相反取向的伙伴存在的情况下，蛋白质才会形成平行的二聚体。相反定向蛋白的共表达几乎完全产生反平行二聚体。最后，平行二聚体可以通过加热洗涤剂溶解的蛋白质而被破坏并转化为反平行二聚体。重要的是，体内功能与反平行二聚体的存在明显相关。我们的结果表明，二聚体中亚单位的反平行排列比平行组织更稳定，可能与蛋白质的功能形式相对应。
The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.