Identification of Novel Functionally Important Aromatic Residue

May 28, 2018 - Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QH, U.K.. ABSTRACT: The extracellular domains ...
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Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Identification of Novel Functionally Important Aromatic Residue Interactions in the Extracellular Domain of the Glycine Receptor Bijun Tang, Steven O. Devenish, and Sarah C. R. Lummis*

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Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QH, U.K. ABSTRACT: The extracellular domains (ECDs) of Cys-loop receptors contain many aromatic amino acids, but only relatively few have been well studied. Here we explore the roles of Tyr and Trp residues in the ECD of the glycine receptor and show that four such residues that have not been previously studied (Y24, Y58, W170, and Y197) contribute significantly to the function of the protein. The residues were studied by creating mutant receptors, characterizing them using two-electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using structural information about the open and closed states of the receptor. Alanine substitution of all these residues ablates function or increases the glycine EC50. There are also a number of changes in the relative maximal responses to taurine, a partial agonist, compared to glycine. Further mutations, in combination with structural information, suggest Y24 contributes to an anion−π interaction with a binding loop D residue, Y58 to an S−π interaction stabilizing the Cys loop, W170 to hydrophobic interactions stabilizing the hydrophobic interior of the subunit, and Y197 to a hydrogen bond linking binding loops B and C. These interactions appear to be broadly conserved in other Cys-loop receptors. Thus, we have identified new regions of the glycine receptor that are important contributors to receptor activation and are likely also to contribute to function in other members of this important protein family.

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the subunit interface and is formed by three segments from one subunit (loops A−C) and three from the adjacent subunit (loops D−F). These loops have a high proportion of aromatic residues, a number of which form the “aromatic box” characteristic of pLGIC orthosteric binding sites. There are also many other aromatic residues in the ECD, some of which have been studied, but most of which have not. As we now have good structural data, we are in an excellent position to visualize how these residues may interact with their neighbors and how such interactions may differ in the open and closed states of the receptor, thus providing information that will ultimately allow us to understand the precise molecular changes that occur when these proteins transform from a resting state to an activated state. Here we probe Trp and Tyr residues in the ECD of homomeric GlyRα1. There are 11 of these aromatic residues in each subunit, and four have been previously studied: Y161 and Y202 are important for agonist binding,13 while the interaction between W68 and W94 stabilizes the top of the ECD and is critical for expression and function.9 Here we determine the roles of the other Tyr and Trp residues (Figures 1 and 2).

lycine receptors (GlyRs), like nicotinic acetylcholine (nACh), GABAA, and 5-HT3 receptors, are members of the pentameric ligand-gated ion channel (pLGIC) family.1−3 They are comprised of five subunits surrounding a central ionconducting pore and can function as homomers or, more commonly, heteromers; four GlyR α subunits and one GlyR β subunit have been identified. Each subunit has a large Nterminal extracellular domain (ECD), a transmembrane domain (TMD) predominantly comprising four α-helices, and an intracellular domain. Binding of glycine to the orthosteric (agonist) binding site, which is located between two adjacent subunits in the ECD, opens an anion-selective channel, and thus activation is inhibitory, with major roles of the GlyR in inhibitory neuronal pathways in the brain stem and spinal cord. High-resolution structural details of full length pLGIC first emerged from prokaryotes;4−6 however, more recently, data from a number of vertebrate pLGICs been published, and in particular, there have been a range of studies of the GlyR.7,8 The GlyRs in these structures are in both bound and unbound forms and thus likely provide a good representation of open and closed states of the receptor. These data have provided support for many previous mutagenesis studies, which have identified residues that are important for the correct assembly, stability, and function of these receptors.9−14 Studies of the binding pocket have been especially extensive. They have shown that the orthosteric (agonist) binding site is located at © XXXX American Chemical Society

Special Issue: Molecules and the Brain Received: April 11, 2018 Revised: May 28, 2018

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DOI: 10.1021/acs.biochem.8b00425 Biochemistry XXXX, XXX, XXX−XXX

Biochemistry



Article

EXPERIMENTAL PROCEDURES

Oocyte Maintenance. Xenopus laevis oocyte-positive females were purchased from NASCO (Fort Atkinson, WI) and maintained according to standard methods. Harvested stage V−VI Xenopus oocytes were washed in four changes of Ca2+-free ND96 [96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES (pH 7.5)], defolliculated in 1.5 mg mL−1 collagenase type 1A for approximately 2 h, washed again in four changes of ND96, and stored in ND96 containing 2.5 mM sodium pyruvate, 50 mM gentamycin, and 0.7 mM theophylline. Receptor Expression. cDNA was cloned into pGEMHE for oocyte expression. Mutagenesis was performed using QuikChange (Agilent Technologies Inc., Santa Clara, CA). cRNA was in vitro transcribed from the linearized pGEMHE cDNA template using the mMessage mMachine T7 tran-

Figure 1. ECD of a GlyR subunit showing the location of Trp and Tyr residues examined in this study.

Figure 2. Clustal alignment of a range of Cys-loop receptor subunit ECDs showing the Tyr and Trp residues examined in this study (red) and those that have been previously studied (purple). The residues in gray boxes have chemical properties similar to those of the equivalent residues in other subunits. The approximate positions of structural features and binding loops A−F are also shown. B

DOI: 10.1021/acs.biochem.8b00425 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry scription kit (Ambion, Austin, TX). Oocytes were injected with 50 nL of ∼400 ng μL−1 cRNA, and currents were recorded 1−4 days postinjection. Electrophysiology. Using a Robocyte voltage clamp system (Multichannel systems), Xenopus oocytes were clamped at −60 mV. Currents were recorded at a frequency of 5 kHz and filtered at 1 kHz. Microelectrodes were filled with 3 M KCl. Pipette resistances ranged from 1.0 to 2.0 MΩ. Oocytes in 50 μL of saline in a V-bottom multiwall plate were perfused at a constant rate of 1 mL min−1. Drug was applied via a simple gravity-fed system calibrated to run at the same rate. Extracellular saline contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.4 with NaOH). Analysis and curve fitting were performed using Prism (GraphPad Software, San Diego, CA). Concentration− response data for each oocyte were normalized to the maximum current for that oocyte. Statistical significance was determined using analysis of variance (ANOVA) with a Dunnett’s multiple-comparison post-test. Structures. Protein Data Bank (PDB) entries 5VDH (open GlyR), 5CFB (closed GlyR), 4PIR (5-HT3AR), 4COF (GABAAR), and 3RHW (GluCl) were downloaded from the PDB and viewed and/or mutated using PyMol or SwissPDBViewer. The GlyR structures are those of the homomeric α3 GlyR, but the majority of residues, and in particular all the aromatic residues studied here, are identical to those in the α1 GlyR. We estimate errors for distances between atoms shown for 5VDH-derived (2.85 Å resolution) and 5CFB-derived (3.04 Å resolution) structures would be 0.2−0.3 Å.15

Figure 3. Typical responses from wild-type (WT), Y58F, and W170A GlyRs. The left panels show the effects of applying a range of concentrations of glycine (WT, 10, 30, 100, 300, and 1000 μM; Y58F, 10, 30, 100, and 300 μM; W170A, 100, 300, 1000, and 3000 μM); the right panels show responses to maximal concentrations of glycine (black) and taurine (red). Scale bars are 2 μA and 20 s.



RESULTS AND DISCUSSION To probe the previously unexplored Trp and Tyr residues in the GlyR ECD, we mutated each to Ala and determined changes in functional characteristics following expression in Xenopus oocytes. We observed a range of changes to EC50. As EC50 = KA(1 + E), where E is the the equilibrium constant for gating or efficacy, it is a function of both binding and gating. Values of KA and E can be obtained from single-channel data, but a less precise qualitative estimate of E can be obtained by comparing the maximal peak current of a partial versus a full agonist, assuming the number of receptors, single-channel conductance, and desensitization rate remain constant.2 We therefore determined the relative maximal response of the partial agonist taurine in our mutant receptors. Typical responses are shown in Figure 3, with concentration−response curves from the most interesting mutant receptors shown in Figure 4. The data from individual residues are discussed in more detail below. Y24. Substitution of Y24 with Ala causes a 3-fold increase in EC50, while substitution with Phe results in a wild-type (WT) EC50 (Table 1), indicating the aromatic group of this residue plays a role in correct receptor function. Y24 is located at the C-terminal end of the N-terminal α-helix at the top of the receptor (Figure 1). The structure of the receptor in this region shows that Y24 is close to D70, which is part of the β2 helix, and to R72 in the β2−β3 loop and thus has the potential to interact with these residues (Figure 5). An anion−π interaction or a cation−π interaction is the most likely, as the WT-like characteristics of the Y24F mutant GlyR indicate a hydrogen bond is not critical here. To test this, we substituted each of these residues with Ala, and the data support an anion−π interaction with D70, as the D70A GlyR had an EC50

Figure 4. Concentration−response curves from WT and mutant GlyRs. Parameters obtained from these data are listed in Table 1.

similar to the values of the forms containing the Y24A mutation (Table 1); the R72A GlyR had an EC50 slightly lower than that of the WT GlyR. There is also the possibility that there is a hydrogen bond between D70 and Y24, as predicted by the structure, which is effectively substituted with an anion−π interaction in the Y24F GlyR Interactions between the N-terminal α-helix and the β2−β3 loop have previously been shown to be important for receptor assembly: alterations to residues in the nACh and 5-HT3 receptor in this region decrease the level of receptor expression, possibly by inhibiting pentamerization of receptors.16,17 It has also been proposed that this region is important for receptor function, e.g., by Lou et al.,18 whose data showing large changes in sensitivity to ACh support conformational changes in this region associated with receptor activation. Our data suggest yet another role of the residues in this region, maintaining the appropriate orientation of residues that are important for agonist binding. D70 is part of loop D, and C

DOI: 10.1021/acs.biochem.8b00425 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Table 1. WT and Mutant GlyR Parametersa pEC50 (M) WT Y24A Y24F Y58A Y58F D70A R72A Y75A Y78A Y128A W170A W170F Y197A Y197F

4.31 ± 0.04 3.73 ± 0.02b 4.20 ± 0.05 3.16 ± 0.04b 4.38 ± 0.01 3.69 ± 0.04b 4.48 ± 0.03b 4.27 ± 0.02 4.29 ± 0.07 4.26 ± 0.03 3.43 ± 0.04b 4.09 ± 0.05 nonresponsive 3.98 ± 0.02b

EC50 (μM)

nH ± ± ± ± ± ± ± ± ± ± ± ±

%Imax Tau/Imax Gly

49 185 63 686 41 201 33 53 51 54 373 81

2.0 1.8 2.5 1.3 1.7 2.7 1.8 2.1 2.2 1.7 2.4 3.0

0.3 0.4 0.2 0.3 0.4 0.4 0.3 0.2 0.3 0.2 0.6 0.4

104

2.5 ± 0.5

51 42 57 151 60 34 91

± ± ± ± ± ± ±

7 5 7 11b 8 7 12b

57 71 129 78

± ± ± ±

5 9 17b 11

35 ± 7

Data are means ± the standard error of the mean (n = 4−8). b Significantly different from the WT values (p < 0.05 via ANOVA with Dunnett’s multiple-comparison post-test). a

Figure 6. Interactions of Y24 (and equivalent) with D70 (or equivalent) in Gly, 5-HT3, and GABAA receptors. Distances and positions of Y24 and D70 in (A) open and (B) closed states of the GlyR. (C) Distance between Y50 and D96 in the 5-HT3A receptor structure. (D) Distance and position between Y23 and D69 in the GABAA receptor structure.

Figure 5. Y24 is