Microbiology and Wine Research, Institute for Molecular Physiology, Johannes Gutenberg University Mainz, Mainz, Germany.
Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany.
J Biol Chem. 2021 Jan-Jun;296:100148. doi: 10.1074/jbc.RA120.015999. Epub 2020 Dec 10.
Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An SxxxGxxxG motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PAS. PAS has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PAS contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PAS (α6) domain across the membrane to α1(PAS). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PAS and kinase regulation.
跨膜（TM）信号转导是膜结合传感器激酶的关键过程。大肠杆菌的 C-二羧酸（延胡索酸）响应传感器激酶 DcuS 通过 TM 螺旋 TM1 和 TM2 锚定在膜中。跨膜信号传输依赖于 TM2 周质部分的活塞式运动。为了定义 TM2 在 TM 信号转导中的作用，我们使用氧化半胱氨酸交联来证明 TM2 在 DcuS 的非激活和延胡索酸激活状态下延伸穿过膜的全长，并形成稳定的 TM 同源二聚体。SxxxGxxxG 基序是 TM2 同源二聚体稳定性和功能所必需的。TM2 螺旋在周质侧进一步延伸到感应 PASP 结构域的α6-螺旋，在细胞质侧延伸到 PAS 的α1-螺旋。PAS 必须将信号传递到 C 端激酶结构域。连接 TM2 和 PAS 的细胞质侧的螺旋接头包含 LxxxLxxxL 序列。在 DcuS 的延胡索酸激活过程中，接头的二聚体状态得到缓解，表明接头发生了结构重排。因此，DcuS 包含一个从感应 PAS（α6）结构域穿过膜延伸到α1（PAS）的长α-螺旋结构。总之，结果表明 TM2 同源二聚体通过 PASP 从 TM 全区域进行活塞式 TM 信号转导，而延胡索酸去稳定化的接头二聚体将信号转换为 PAS 和激酶调节的细胞质侧。
