Hunger Doreen, Röcker Marie, Falke Dörte, Lilie Hauke, Sawers R Gary
Institute of Microbiology, Martin-Luther University Halle-WittenbergHalle, Germany.
Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-WittenbergHalle, Germany.
Front Microbiol. 2017 Aug 22;8:1616. doi: 10.3389/fmicb.2017.01616. eCollection 2017.
FocA is the archetype of the pentameric formate-nitrite transporter (FNT) superfamily of channels, members of which translocate small organic and inorganic anions across the cytoplasmic membrane of microorganisms. The N- and C-termini of each protomer are cytoplasmically oriented. A Y-L-R motif is found immediately after transmembrane helix 6 at the C-terminus of FNT proteins related to FocA, or those with a role in formate translocation. Previous studies had revealed that formate translocation through FocA was controlled by interaction with the formate-producing glycyl-radical enzyme pyruvate formate-lyase (PflB) or its structural and functional homolog, TdcE. In this study we analyzed the effect on formate export and import, as well as on the stability of the homopentamer in the membrane, of successively removing amino acid residues from the C-terminus of FocA. Removal of up to five amino acids was without consequence for either formate translocation or oligomer stability. Removal of a sixth residue (R280) prevented formate uptake by FocA in a strain lacking PflB and significantly reduced, but did not prevent, formate export. Sensitivity to the toxic formate analog hypophosphite, which is also transported into the cell by FocA, was also relieved. Circular dichroism spectroscopy and blue-native PAGE analysis revealed, however, that this variant had near identical secondary and quaternary structural properties to those of native FocA. Interaction with the glycyl radical enzyme, TdcE, was also unaffected by removal of the C-terminal 6 amino acid residues, indicating that impaired interaction with TdcE was not the reason for impaired formate translocation. Removal of a further residue (L279) severely restricted formate export, the stability of the protein and its ability to form homopentamers. Together, these studies revealed that the Y-L-R motif at the C-terminus is essential for bidirectional formate translocation by FocA, but that L279 is both necessary and sufficient for homopentamer integrity.
FocA是五聚体型甲酸-亚硝酸盐转运蛋白（FNT）通道超家族的原型，该超家族成员可使小的有机和无机阴离子穿过微生物的细胞质膜。每个亚基的N端和C端都朝向细胞质。在与FocA相关的FNT蛋白的C端跨膜螺旋6之后，或者在那些在甲酸转运中起作用的蛋白的C端跨膜螺旋6之后，发现了一个Y-L-R基序。先前的研究表明，通过FocA的甲酸转运受与产生甲酸的甘氨酰自由基酶丙酮酸甲酸裂解酶（PflB）或其结构和功能同源物TdcE的相互作用控制。在本研究中，我们分析了从FocA的C端连续去除氨基酸残基对甲酸输出和输入以及对膜中同五聚体稳定性的影响。去除多达五个氨基酸对甲酸转运或寡聚体稳定性均无影响。去除第六个残基（R280）可阻止缺乏PflB的菌株中FocA对甲酸的摄取，并显著降低但并未阻止甲酸的输出。对毒性甲酸类似物次磷酸盐的敏感性也有所缓解，次磷酸盐也是通过FocA转运进入细胞的。然而，圆二色光谱和蓝色非变性聚丙烯酰胺凝胶电泳分析表明，该变体的二级和四级结构特性与天然FocA几乎相同。与甘氨酰自由基酶TdcE的相互作用也不受C端6个氨基酸残基去除的影响，这表明与TdcE相互作用受损不是甲酸转运受损的原因。再去除一个残基（L279）会严重限制甲酸输出、蛋白质的稳定性及其形成同五聚体的能力。总之，这些研究表明，C端的Y-L-R基序对于FocA双向转运甲酸至关重要，但L279对于同五聚体的完整性既是必要的也是充分的。
