Formate translocation by the FocA channel: How the N-terminal domain modulates anion specificity

在pH > 6.5下生长的发酵肠细菌将甲酸从细胞质（其中甲酸由丙酮酸甲酸裂解酶（PflB）产生）转移到周质。在pH < 6.5时，甲酸盐被吸收回细胞中，在细胞中甲酸盐被甲酸氢裂解酶复合物还原为CO2和H2。通过福卡进行甲酸盐穿过细胞质膜的可逆易位，福卡属于进化上古老的甲酸盐-亚硝酸盐转运蛋白（FNT）家族，其成员是显示出与四聚体水通道蛋白的结构相似性的150 kDa五聚体通道。福卡的每个原聚体都有一个疏水孔，在体内可逆地易位甲酸;这是如何实现的，以及通道的“门控”如何发生，还没有完全理解。调查这一易位过程是本建议的重点。福卡的疏水孔含有位于中心的组氨酸残基（H209），其是孔中唯一带电荷的氨基酸。该组氨酸残基在大约99%的所有FNT蛋白中是保守的。唯一已知的例外是在这个位置上有天冬酰胺或谷氨酰胺。我们最近的研究结果表明，当H209转化为这些非质子化的酰胺残基，福卡成为一个排他性的甲酸流出通道。这表明H209是福卡A摄取甲酸所必需的，并提示了大肠杆菌中pH依赖性甲酸转运的生理学原理。杆菌氨基酸T91在疏水孔内也是高度保守的，并且其位于H209附近。T91对于允许可逆阴离子渗透通过孔也是必不可少的。我们最近确定了完整的福卡通道的3.1 μ m结构（与P. Kapleman在Halle的合作），使用冷冻EM，这支持了H209和T91在控制阴离子通道中的作用。由于几乎所有的FNT通道在每个原聚体的疏水孔内具有许多保守的残基，这表明阴离子特异性是通过另一种机制实现的。我们最近发现，位于细胞质中的福卡A的N-末端结构域对其体内功能至关重要。此外，我们的数据表明，PfIB特异性结合到这个N-末端结构域，这表明这种相互作用可能控制阴离子进入易位孔。不同FNT的N-末端结构域在序列或二级结构上没有显示出保守性。这表明通过N-末端结构域控制FNT孔门控可能代表跨越整个超家族的共同机制。我们计划用三种不同的FNT进行结构域交换实验来验证这一假设。因此，本提案的总体目标是阐明保守的孔残基在甲酸盐易位中的功能作用，并确定N-末端结构域如何控制可逆的甲酸盐通过福卡孔。

Fermenting enterobacteria growing at pH > 6.5 translocate formate from the cytoplasm, where it is generated by pyruvate formate-lyase (PflB), to the periplasm. At pH < 6.5, formate is taken back up into the cell, where it is disproportionated to CO2 and H2 by the formate hydrogenlyase complex. Reversible translocation of formate across the cytoplasmic membrane is carried out by FocA, which belongs to the evolutionarily ancient formate-nitrite transporter (FNT) family, members of which are 150 kDa pentameric channels that show structural similarity to tetrameric aquaporins. Each protomer of FocA has a hydrophobic pore that in vivo reversibly translocates formate; how this is achieved, and how ‘gating’ of the channel occurs, are not completely understood. Investigation of this translocation process is the focus of this proposal. The hydrophobic pore of FocA contains a centrally localized histidine residue (H209), which is the only charged amino acid in the pore. This histidine residue is conserved in roughly 99% of all FNT proteins. The only known exceptions have either asparagine or glutamine at this position. Our recent findings indicate that when H209 is converted to either of these non-protonable amide residues, FocA becomes an exclusive formate efflux channel. This indicates H209 is necessary for formate uptake by FocA and suggests a physiological rationale for pH-dependent formate translocation in E. coli. Amino acid T91 is also highly conserved within the hydrophobic pore and it is located in proximity to H209. T91 is also essential to allow reversible anion permeation through the pore. We have recently determined the 3.1 Å structure of the complete FocA channel (collaboration with P. Kastritis in Halle) using cryo-EM and this supports a role for H209 and T91 in controlling anion passage. As nearly all FNT channels have a number of conserved residues within the hydrophobic pore of each protomer, this suggests anion specificity is achieved by another mechanism. We have shown recently that the cytoplasmically localized N-terminal domain of FocA is essential for its in vivo function. Moreover, our data indicate that PflB specifically binds to this N-terminal domain, suggesting this interaction might control anion access to the translocation pore. The N-terminal domains of different FNTs show no conservation in sequence or secondary structure. This suggests that control of FNT pore gating by the N-terminal domain might represent a common mechanism spanning the complete superfamily. We plan domain-swapping experiments with three different FNTs to test this hypothesis. Thus, the overall aims of this proposal are to elucidate the functional roles of conserved pore residues in formate translocation and to determine how the N-terminal domain acts to control reversible formate passage through the FocA pore.