Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase

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 PAS_C. PAS_C has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PAS_C 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_P (α6) domain across the membrane to α1(PAS_C). 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_C and kinase regulation.