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.
Highlights
Neurotransmitter receptors are vital signaling proteins that are embedded in the cell membrane and trigger intracellular changes in response to extracellular chemical signals
These results suggest that the positive charge of Arg76 contributes little to glutamate binding in GluCls
Our results show that two other aspects of the Arg side chain contribute to effective glutamate recognition
Summary
To test whether positive charge of Arg residues 76 and 95 is sufficient for glutamate recognition in the glutamate binding site of the AVR-14B GluCl, we replaced these individually with Lys via site-directed mutagenesis and measured glutamategated chloride currents with two electrode voltage clamp experiments (Fig. 1, A and B). Arg-to-Lys substitution largely retains side chain size and charge, it involves the loss of two potentially hydrogen-bonding (H-bonding) nitrogen atoms (Fig. 2A), which could underlie the loss of glutamate sensitivity we observed in R76K and R95K mutants. Assessing the precise contributions of charge or H-bonding of the Arg side chain to glutamate recognition would require substitution with an uncharged but otherwise isosteric analog. To this end, we sought to replace Arg or Arg with canavanine (Can), an isosteric Arg analog with a pKa of 7 [15] (Fig. 2A), reasoning that experiments at low and high pH would assay glutamate sensitivity when a single guanidino side chain is protonated or deprotonated, respectively. For comparison with Can (oocytes injected with 40 ng of UAG mutant mRNA and tRNA-Can; Fig. 2), oocytes were injected with only 0.04 ng of Arg WT mRNA, to keep WT expression levels low, and Imax values comparable with Can76. ***, p Ͻ 0.001 as compared with pH 7.0 (ANOVA with Tukey’s post test)
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