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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. IMPORTANCE Here, we studied the LytS-type histidine kinase BtsS of E. coli and identified the pyruvate binding site within the membrane-spanning domains. It is a small cavity, and pyruvate forms interactions with the side chains of Arg72, Arg99, Cys110, and Ser113 located in transmembrane helices III, IV, and V, respectively. Our results can serve as a starting point to convert BtsS into a sensor for structurally similar ligands such as lactate, which can be used as biosensor in medicine.