Abstract
Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-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 S186xxxGxxxG194 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 PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC 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 PASP (α6) domain across the membrane to α1(PASC). 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 PASC and kinase regulation.
Highlights
Membrane-anchored bacterial histidine kinases typically perceive ambient stimuli via their extra-cytoplasmic sensor domains [1, 2]
The fumarate, or C4-dicarboxylate (C4DC), sensor kinase DcuS of the DcuS–DcuR two-component system consists of a PASP (Per-ARNT-SIM) sensor domain, a TM domain composed of the antiparallel transmembrane helices TM1 and TM2, and a cytoplasmic PASC and the kinase domain [22, 23]
We show that DcuS contains a long continuous α-helix connecting the periplasmic PASP sensor domain with PASC on the cytoplasmic side of the membrane
Summary
TM2 of DcuS homodimerizes at specific contact sites regardless of activity state. The role of TM2 and the adjacent cytoplasmic region in DcuS homodimerization was analyzed in aerobically grown Escherichia coli cells, i.e., in the native context with DctA, by Cys CL using membrane-permeant copper(II)-(1,10phenanthroline), or “Cu2+ phenanthroline,” as an oxidant [18, 32, 33]. The Cys variants retained all 87%–118% of DcuSCys0 activity in dcuB-lacZ expression and induced dcuB-lacZ in a fumarate-dependent manner as the wild-type (Fig. 2). Ser182 to Leu201), the periplasmic α6 of PASP, and the cytoplasmic α1-PASC helices (see Fig. 1B). A +3/+4 periodicity of high CL efficiency was observed (Fig. 1B), which is characteristic of the interaction between two α-helices. The clearest differences were observed in the Cys199–Lys207 region, corresponding to the TM2-PASC interdomain or linker region. In this region, a generally low CL efficiency was observed with partial nonhelical spacing of the CL maxima, and relatively large fumarate-induced differences, especially for residues Leu201, Val202, Val204, Leu205, and Lys206. The CL results suggest that the region from α6 in PASP to α1 in PASC forms a continuous α-helix, including the complete TM2 region
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