Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor
Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor
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DOI：
10.1128/jb.01710-07
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发表时间：
2008-05-01
影响因子：
3.2
通讯作者：
Narnba, Kelichi
中科院分区：
文献类型：
--
作者：
Kojima, Seiji;Furukawa, Yukio;Narnba, Kelichi
MOTA和MOTB是构成质子驱动细菌鞭毛马达定子复合体的完整膜蛋白。定子复合体起到质子通道的作用，并将质子如何与扭矩产生耦合。定子必须锚定到电机上的适当位置，这被认为是通过MOTB的C-末端周质结构域中的一个可能的肽聚糖结合(PGB)基序发生的。在这项研究中，我们构建并鉴定了肠系沙门氏菌MOTB的一个N-末端截短的变种，该变种由78到309个残基组成(MOTB(C))。MOTB(C)在输出到周质时显著抑制野生型细胞的运动。PGB基序的一些点突变增强了对运动的抑制，而框内缺失变体MOTB(C)(Delta 197-210)则显示出显著的抑制作用。野生型MOTB(C)及其点突变突变体形成稳定的同源二聚体，而缺失突变体为单体。镍-氨基三乙酸亲和层析仅与分泌形式的MOTB(C)-His(6)共分离出少量MOTB，提示MOTB-MOTB(C)异二聚体的形成可能是运动抑制的结果。然而，单体突变体MOTB(C)(Delta 197-210)不与MOTB结合，表明MOTB(C)直接参与定子组装。我们认为MOTB(C)二聚体结构域在MOTA/MOTB复合体的靶向和稳定锚定到电机可能的定子结合部位方面起着重要作用。
MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton How with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotB(C)). MotB(C) significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotB(C)(Delta 197-210), showed a significantly reduced inhibitory effect. Wild-type MotB(C) and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotB(C)-His(6) by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB(C) heterodimer formation in the periplasm. However, the monomeric mutant variant MotB(C) (Delta 197-210) did not bind to MotB, suggesting that MotB(C) is directly involved in stator assembly. We propose that the MotB(C) dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.