Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Berlin, Germany.
J Bacteriol. 2012 Sep;194(17):4505-12. doi: 10.1128/JB.00683-12. Epub 2012 Jun 15.
Energy-coupling factor transporters are a large group of importers for trace nutrients in prokaryotes. The in vivo oligomeric state of their substrate-specific transmembrane proteins (S units) is a matter of debate. Here we focus on the S unit BioY of Rhodobacter capsulatus, which functions as a low-affinity biotin transporter in its solitary state. To analyze whether oligomerization is a requirement for function, a tail-to-head-linked BioY dimer was constructed. Monomeric and dimeric BioY conferred comparable biotin uptake activities on recombinant Escherichia coli. Fluorophore-tagged variants of the dimer were shown by fluorescence anisotropy analysis to oligomerize in vivo. Quantitative mass spectrometry identified biotin in the purified proteins at a stoichiometry of 1:2 for the BioY monomer and 1:4 (referring to single BioY domains) for the dimer. Replacement of the conserved Asp164 (by Asn) and Lys167 (by Arg or Gln) in the monomer and in both halves of the dimer inactivated the proteins. The presence of those mutations in one half of the dimers only slightly affected biotin binding but reduced transport activity to 25% (Asp164Asn and Lys167Arg) or 75% (Lys167Gln). Our data (i) suggest that intermolecular interactions of domains from different dimers provide functionality, (ii) confirm an oligomeric architecture of BioY in living cells, and (iii) demonstrate an essential role of the last transmembrane helix in biotin recognition.
能量偶联因子转运蛋白是原核生物中痕量营养物质的一大类进口蛋白。其底物特异性跨膜蛋白（S 单位）的体内寡聚状态仍存在争议。在这里，我们关注的是荚膜红细菌的 S 单位 BioY，它在其单体状态下作为低亲和力生物素转运蛋白发挥作用。为了分析寡聚化是否是功能所必需的，构建了一个从头至尾连接的 BioY 二聚体。单体和二聚体 BioY 赋予重组大肠杆菌相当的生物素摄取活性。荧光各向异性分析表明，荧光标记的二聚体变体在体内发生寡聚化。定量质谱分析在纯化的蛋白质中鉴定出生物素，其单体的生物素与蛋白质的摩尔比为 1:2，二聚体的摩尔比为 1:4（指单个 BioY 结构域）。单体中的保守天冬氨酸 164（突变为天冬酰胺）和赖氨酸 167（突变为精氨酸或谷氨酰胺）以及二聚体的两个半体中的赖氨酸 167（突变为精氨酸或谷氨酰胺）都使蛋白失活。二聚体的一半中的这些突变的存在仅略微影响生物素结合，但将转运活性降低至 25%（天冬氨酸 164 突变为天冬酰胺和赖氨酸 167 突变为精氨酸）或 75%（赖氨酸 167 突变为谷氨酰胺）。我们的数据表明：（i）来自不同二聚体的结构域之间的分子间相互作用提供了功能；（ii）证实了 BioY 在活细胞中的寡聚结构；（iii）证明了最后一个跨膜螺旋在生物素识别中的重要作用。
