The LytS-type histidine kinase BtsS is a 7-transmembrane receptor that binds pyruvate

Abstract

LytS/LytTR-type histidine kinase/response regulator systems regulate crucial host-specific mechanisms during infection of human or plant hosts. Escherichia coli has two of them, and the histidine kinase BtsS is a high-affinity sensor for extracellular pyruvate that together with the response regulator BtsR activates the expression of btsT, which encodes a high-affinity pyruvate transporter. However, the molecular mechanism of how pyruvate binding triggers a response is still unclear. Here, we experimentally show that BtsS consists of seven transmembrane helices, with the N-terminus exposed on the periplasmic side. Using a site-directed mutagenesis-based screening assay, Arg72, Arg99, Cys110, and Ser113, all located on the periplasmic side in three transmembrane helices, were identified as critical for the binding of pyruvate to BtsS, a finding that was further confirmed by molecular dynamics simulation studies. In addition, Mn2+dependent autophosphorylation of wild-type BtsS was demonstrated for the first time, and individual replacement of the four amino acids affected this process as well as dimerization and consequently btsT expression. This study demonstrates how binding of a metabolite to the membrane-integrated sensor domain triggers signaling in the cytoplasm.