DIVALENT METAL CATIONS ARE INVOLVED IN LIGAND RECOGNITION BY THE CITRATE CHEMORECEPTOR TCP OF SALMONELLA ENTERICA

The chemoreceptor Tcp of Salmonella enterica is unique in that it mediates attractant responses not only to citrate but also to divalent metal cations in the presence of citrate but not in its absence. Tcp has therefore been supposed to sense citrate and metal-bound citrate as distinct attractants. In collaboration with this laboratory, Dr. Katsumi Imada of Osaka University determined the crystal structure of a periplasmic fragment of Tcp. In each ligand-binding pocket of the Tcp homodimer, a Zn^2+ ion was accommodated with a citrate molecule. In this study, I aimed at understanding the molecular mechanism of ligand recognition by Tcp with particular interest in the role of divalent cations. First, I found that EDTA dramatically decreased citrate response of the receptor-less Escherichia coli strain expressing Tcp as a sole chemoreceptor, and that this inhibition was reversed by the addition of Mg^2+. Effects of EDTA and Mg^2+ on citrate responses of cells expressing several mutant Tcp proteins further support the notion that Tcp requires divalent cations for citrate recognition. Next, I purified a periplasmic fragment of Tcp to carry out isothermal titration calorimetry (ITC) measurements. However, binding of citrate was not detected presumably because the fragment was not stable. I then tried to construct more stable Tcp fragments. Out of 6 fragments constructed, the fragment spanning Thr-24 to Ser-186 was less degraded, and the introduction of the D35C mutation that promotes dimerization seemed to enhance the stability