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J Biol Chem. 2017 Mar 3;292(9):3940-3946. doi: 10.1074/jbc.M116.772939. Epub 2017 Jan 17.

Unique Contributions of an Arginine Side Chain to Ligand Recognition in a Glutamate-gated Chloride Channel.

Author information

1
From the Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, 2100 H Copenhagen, Denmark.
2
From the Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, 2100 H Copenhagen, Denmark stephan.pless@sund.ku.dk.

Abstract

Glutamate recognition by neurotransmitter receptors often relies on Arg residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function and pharmacology has proven to be exceedingly difficult in such large and complex proteins. Using the in vivo nonsense suppression approach, we report the first successful incorporation of the isosteric, titratable Arg analog, canavanine, into a neurotransmitter receptor in a living cell, utilizing a glutamate-gated chloride channel from the nematode Haemonchus contortus Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via a hydrogen bond network, where Arg interacts both with agonist and with a conserved Thr side chain within the receptor. Together, the data provide a new explanation for the reliance of neurotransmitter receptors on Arg side chains and highlight the exceptional capacity of unnatural amino acid incorporation for increasing our understanding of ligand recognition.

KEYWORDS:

Unnatural amino acids; arginine; biophysics; channel activation; glutamate; ion channel; ligand recognition; membrane protein; neurotransmitter

PMID:
28096462
PMCID:
PMC5339774
DOI:
10.1074/jbc.M116.772939
[Indexed for MEDLINE]
Free PMC Article

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