Structure–Activity Relationship Study Reveals the Molecular Basis for Specific Sensing of Hydrophobic Amino Acids by the Campylobacter jejuni Chemoreceptor Tlp3

Abstract

Chemotaxis is an important virulence factor of the foodborne pathogen Campylobacter jejuni. Inactivation of chemoreceptor Tlp3 reduces the ability of C. jejuni to invade human and chicken cells and to colonise the jejunal mucosa of mice. Knowledge of the structure of the ligand-binding domain (LBD) of Tlp3 in complex with its ligands is essential for a full understanding of the molecular recognition underpinning chemotaxis. To date, the only structure in complex with a signal molecule is Tlp3 LBD bound to isoleucine. Here, we used in vitro and in silico screening to identify eight additional small molecules that signal through Tlp3 as attractants by directly binding to its LBD, and determined the crystal structures of their complexes. All new ligands (leucine, valine, α-amino-N-valeric acid, 4-methylisoleucine, β-methylnorleucine, 3-methylisoleucine, alanine, and phenylalanine) are nonpolar amino acids chemically and structurally similar to isoleucine. X-ray crystallographic analysis revealed the hydrophobic side-chain binding pocket and conserved protein residues that interact with the ammonium and carboxylate groups of the ligands determine the specificity of this chemoreceptor. The uptake of hydrophobic amino acids plays an important role in intestinal colonisation by C. jejuni, and our study suggests that C. jejuni seeks out hydrophobic amino acids using chemotaxis.

The role of solute binding proteins in signal transduction

“…Cache domains are the most abundant extracytosolic bacterial sensor domains [13] , [14] , [62] and the majority of Cache domains are present in their dCache configuration. A large number of dCache domains have been studied, and, in almost all cases, ligands bind to the membrane-distal module [63] , [64] , [65] , [66] . That circumstance raised the question as to the function of the membrane-proximal module.…”

The Signaling Pathway That cGAMP Riboswitches Found: Analysis and Application of Riboswitches to Study cGAMP Signaling in Geobacter sulfurreducens

“…Cache domains are small molecule-binding domains commonly associated with signaling enzymes and chemotaxis receptors. Several Cache domains from methyl chemotaxis proteins have been found to bind amino acids, including ones with solved x-ray crystal structures such as PctA from Pseudomonas aeruginosa that binds most amino acids [26], Mlp24 from Vibrio cholerae that binds serine, alanine, and arginine [27], and Tlp3 from Campylobacteri jejuni that binds nonpolar amino acids [28].…”

Histamine: A Bacterial Signal Molecule

“…Like PctA and PctC, the TlpQ chemoreceptor has a dCache type LBD, and its 3D structure in a complex with histamine has been solved by X-ray crystallography [23] (Figure 2). The TlpQ-LBD is composed of two structural α/β modules, and histamine was bound at the membrane distal module, like in the very large majority of other characterized dCache domains [59,[65][66][67][68]. The molecular detail of histamine recognition by human receptors has recently been deciphered by reporting three dimensional structures of the Histamine H1 receptor [69] and the β3 GABA A receptor in a complex with histamine [70] (Figure 2).…”