There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two‐component systems from marine Shewanella species, and validate them in laboratory Escherichia coli. Then, we port these sensors into a gut‐adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.
The recently published crystal structure of the D3 dopamine receptor shows a tightly packed region of aromatic residues on helices 5 and 6 in the space bridging the binding site and what is thought to be the origin of intracellular helical motion. This highly conserved region also makes contacts with residues on helix 3, and here we use double mutant cycle analysis and unnatural amino acid mutagenesis to probe the functional role of several residues in this region of the closely related D2 dopamine receptor. Of the eight mutant pairs examined, all show significant functional coupling (Ω > 2), with the largest coupling coefficients observed between residues on different helices, C3.36/W6.48, T3.37/S5.46 and F5.47/F6.52. Additionally, three aromatic residues examined, F5.47, Y5.48, and F5.51 show consistent trends upon progressive fluorination of the aromatic side chain. These trends are indicative of a functionally important electrostatic interaction with the face of the aromatic residue examined, which is likely attributed to aromatic-aromatic interactions between residues in this microdomain. We also propose that the previously determined fluorination trend at W6.48 is likely due to a sulfur-π interaction with the side chain of C3.36. We conclude that these residues form a tightly packed structural microdomain that connects helices 3, 5, and 6, thus forming a barrier that prevents dopamine from binding further toward the intracellular surface. Upon activation, these residues likely do not change their relative conformation, but rather act to translate agonist binding at the extracellular surface into the large intracellular movements that characterize receptor activation.
publication of the first high-resolution crystal structure
of a eukaryotic Cys-loop receptor, GluClα, has provided valuable
structural information on this important class of ligand-gated ion
channels (LGIC). However, limited functional data exist for the GluCl
receptors. Before applying the structural insights from GluCl to mammalian
Cys-loop receptors such as nicotinic acetylcholine and GABA receptors,
it is important to ensure that established functional features of mammalian Cys-loop receptors are present in the more
distantly related GluCl receptors. Here, we seek to identify ligand-binding
interactions that are generally associated with Cys-loop receptors,
including the frequently observed cation−π interaction.
Our studies were performed on the highly homologous GluClβ receptor,
because GluClα is not activated by glutamate in Xenopus
laevis oocytes. Mutagenesis of the signal peptide and pore
lining helix was performed to enhance functional expression and sensitivity
to applied ligand, respectively. Conventional and unnatural amino
acid mutagenesis indicate a strong cation−π interaction
between Y206 and the protonated amine of glutamate, as well as other
important ionic and hydrogen bond interactions between the ligand
and the binding site, consistent with the crystal structure.
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