The chemoreceptor Tcp mediates taxis to citrate. To identify citrate-binding residues, we substituted cysteine for seven basic or polar residues that are chosen based on the comparison of Tcp with the well-characterized chemoreceptors. The results suggest that Arg-63, Arg-68, Arg-72, Lys-75, and Tyr-150 (and probably other unidentified residues) are involved in the recognition of citrate.The closely related enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium have multiple transmembrane receptors that mediate chemotactic responses to amino acids, non-PTS sugars, and other attractants and repellents (2,11,25,26,34,35). Some receptors (Tar for aspartate, Tsr for serine, and Trg for ribose and galactose) are found in both species. Others are species specific. Belonging to the latter class is the Salmonella serovar Typhimurium-specific chemoreceptor Tcp, which mediates taxis to citrate and a divalent cation-citrate complex and away from phenol (39). Salmonella serovar Typhimurium, but not E. coli, can utilize citrate as a sole carbon source.Ligand recognition by Tar and Tsr has been studied extensively using mutagenesis (13, 21, 22, 30, 38), chemical modification (14, 15, 17), X-ray crystallography (28, 40), and computer simulation (19). Mutations at the aspartate-binding residues of Tar cause defects in the aspartate-sensing ability without affecting the repellent-sensing ability or other receptor functions (13,21,30,38), although in some cases the maltosesensing ability is also affected due to a partial overlap of the binding sites for aspartate and the complex of maltose and maltose-binding protein (13). Tcp is homologous to Tar and Tsr, but it recognizes the non-amino acid ligand citrate. Identification of the citrate-binding residues of Tcp should further our understanding of the molecular logic underlying ligand recognition in this family of receptors.The residues involved in ligand binding in Tar and Tsr ( Fig. 1A and B) are located in the two helices (␣1 and ␣4) that extend through the cytoplasmic membrane as the first and the second transmembrane regions (TM1 and TM2), respectively (28, 40). In Tar, three Arg residues (residues 64, 69, and 73) within the helix ␣1 interact with the ␣-or ␤-carboxyl groups of aspartate (40) (Fig. 1A and B). The corresponding Arg residues of Tsr are predicted to interact with the ␣-carboxyl or the hydroxyl group of serine (19,22) (Fig. 1A and B). This triplet is perfectly conserved in Tcp as residues 63, 68, and 72 (Fig. 1B), which are expected to interact with the carboxyl groups or the hydroxyl group of citrate. Tyr-149 of Tar, which is located near the top of the helix ␣4 and interacts with the carboxyl groups of the ligand via water molecules (40), is also conserved in Tcp as Tyr-150. Because citrate has no amino group (Fig. 1C), it is reasonable that the Thr residue (154 in Tar and 156 in Tsr), which interacts with the amino group of aspartate or serine, is not conserved in Tcp (39). Since citrate has a polar hydroxyl group and three carboxyl gr...