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Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli.

ABSTRACT: Umami taste perception is mediated by the heterodimeric G-protein coupled receptors (GPCRs), formed by the assembly of T1R1 and T1R3 subunits. T1R1 and T1R3 subunits are class C GPCRs whose members share common structural homologies including a long N-terminal domain (NTD) linked to a seven transmembrane domain by a short cysteine-rich region. The NTD of the T1R1 subunit contains the primary binding site for umami stimuli, such as L-glutamate (L-Glu) for humans. Inosine-5'-monophosphate (IMP) binds at a location close to the opening of the T1R1-NTD "flytrap", thus creating the observed synergistic response between L-Glu and IMP. T1R1/T1R3 binding studies have revealed species-dependent differences. While human T1R1/T1R3 is activated specifically by L-Glu, the T1R1/T1R3 in other species is a broadly tuned receptor, sensitive to a range of L-amino acids. Because domestic cats are obligate carnivores, they display strong preferences for some specific amino acids. To better understand the structural basis of umami stimuli recognition by non-human taste receptors, we measured the binding of selected amino acids to cat T1R1/T1R3 (cT1R1/cT1R3) umami taste receptor. For this purpose, we expressed cT1R1-NTD in bacteria as inclusion bodies. After purification, refolding of the protein was achieved. Circular dichroism spectroscopic studies revealed that cT1R1-NTD was well renatured with evidence of secondary structures. Using size-exclusion chromatography coupled to light scattering, we found that the cT1R1-NTD behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that cT1R1-NTD is capable of binding L-amino acids with Kd values in the micromolar range. We demonstrated that IMP potentiates L-amino acid binding onto renatured cT1R1-NTD. Interestingly, our results revealed that IMP binds the extracellular domain in the absence of L-amino acids. Thus, this study demonstrates that the feasibility to produce milligram quantities of cT1R1-NTD for functional and structural studies.

SUBMITTER: Belloir C

PROVIDER: S-EPMC5662223 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli.

Belloir Christine C Savistchenko Jimmy J Neiers Fabrice F Taylor Andrew J AJ McGrane Scott S Briand Loïc L

PloS one 20171030 10

Umami taste perception is mediated by the heterodimeric G-protein coupled receptors (GPCRs), formed by the assembly of T1R1 and T1R3 subunits. T1R1 and T1R3 subunits are class C GPCRs whose members share common structural homologies including a long N-terminal domain (NTD) linked to a seven transmembrane domain by a short cysteine-rich region. The NTD of the T1R1 subunit contains the primary binding site for umami stimuli, such as L-glutamate (L-Glu) for humans. Inosine-5'-monophosphate (IMP) bi ...[more]

PMID: 29084235

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Project description:UnlabelledCS6 is a common colonization factor expressed by enterotoxigenic Escherichia coli It is a two-subunit protein consisting of CssA and CssB in an equal stoichiometry, assembled via the chaperone-usher pathway into an afimbrial, oligomeric assembly on the bacterial cell surface. A recent structural study has predicted the involvement of the N- and C-terminal regions of the CS6 subunits in its assembly. Here, we identified the functionally important residues in the N- and C-terminal regions of the CssA and CssB subunits during CS6 assembly by alanine scanning mutagenesis. Bacteria expressing mutant proteins were tested for binding with Caco-2 cells, and the results were analyzed with respect to the surface expression of mutant CS6. In this assay, many mutant proteins were not expressed on the surface while some showed reduced expression. It appeared that some, but not all, of the residues in both the N and C termini of CssA and CssB played an important role in the intermolecular interactions between these two structural subunits, as well as chaperone protein CssC. Our results demonstrated that T20, K25, F27, S36, Y143, and V147 were important for the stability of CssA, probably through interaction of CssC. We also found that I22, V29, and I33 of CssA and G154, Y156, L160, V162, F164, and Y165 of CssB were responsible for CssA-CssB intermolecular interactions. In addition, some of the hydrophobic residues in the C terminus of CssA and the N terminus of CssB were involved in the stabilization of higher-order complex formation. Overall, the results presented here might help in understanding the pathway used to assemble CS6 and predict its structure.ImportanceUnlike most other colonization factors, CS6 is nonfimbrial, and in a sense, its subunit composition and assembly are also unique. Here we report that both the N- and C-terminal amino acid residues of CssA and CssB play a critical role in the intermolecular interactions between them and assembly proteins. We found mainly that alternate hydrophobic residues present in these motifs are essential for the interaction between the structural subunits, as well as the chaperone and usher assembly proteins. Our results indicate the involvement of the side chains of identified amino acids in CS6 assembly. This study adds a step toward understanding the interactions between structural subunits of CS6 and assembly proteins during CS6 biogenesis.

Dataset Information

Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli.

Publications

Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli.

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