Engineering a surrogate human heteromeric α/β glycine receptor orthosteric site exploiting the structural homology and stability of acetylcholine-binding protein

Abstract

Protein-engineering methods have been exploited to produce a surrogate system for the extracellular neurotransmitter-binding site of a heteromeric human ligand-gated ion channel, the glycine receptor. This approach circumvents two major issues: the inherent experimental difficulties in working with a membrane-bound ion channel and the complication that a heteromeric assembly is necessary to create a key, physiologically relevant binding site. Residues that form the orthosteric site in a highly stable ortholog, acetylcholine-binding protein, were selected for substitution. Recombinant proteins were prepared and characterized in stepwise fashion exploiting a range of biophysical techniques, including X-ray crystallography, married to the use of selected chemical probes. The decision making and development of the surrogate, which is termed a glycine-binding protein, are described, and comparisons are provided with wild-type and homomeric systems that establish features of molecular recognition in the binding site and the confidence that the system is suited for use in early-stage drug discovery targeting a heteromeric α/β glycine receptor.

Keywords: acetylcholine-binding protein; crystal structures; glycine receptor; ligand-gated ion channel; nicotine; strychnine; tropisetron.

Figure 1

A schematic of a heteropentameric GlyR. The stoichiometry is (α1)₂(β)₃, with the α1 subunit in red and the β subunit in cyan. Plus and minus symbols indicate the positions of the principal and complementary sides of the binding site, respectively. In this arrangement there are three types of binding site: two α1(+)/β(−), two α1(−)/β(+) and one β(+)/β(−). (b) Comparison of the loop segments that create the orthosteric ligand-binding sites in AcAChBP, human GlyR-α1 and GlyR-β. The residues colored red indicate where amino-acid substitutions have been carried out to create GBP. The four residues colored blue contribute to the binding site but have not been changed owing to structural conservation.

Figure 2

Schematic to describe the construction of and key residues in the orthosteric binding site of AcAChBP and the corresponding amino acids in the human GlyR-α1(−)/β(+) heteromeric site. Substitutions in red convert AcAChBP into GBP.

Figure 3

Tropisetron adopts two poses in the orthosteric site of variant II. (a) The chemical structure of tropisetron. (b) The interacting residues of variant II are shown with C positions colored white for the principal side and cyan for the complementary side, with one tropisetron pose (yellow C positions). Two water molecules discussed in the text are depicted as blue spheres; O and N positions are red and blue, respectively. Selected hydrogen-bonding interactions are shown as blue dashed lines. The second pose, which is common with that adopted in WT AcAChBP (PDB entry 2wnc), is shown with black C atoms.

Figure 4

Glycine in an orthosteric site of GBP. A similar color scheme as shown in Fig. 3 ▸ is used, with glycine C positions in black.

Figure 5

Strychnine bound to GBP. (a) The chemical structure of the natural product. (b) The key residues and orientation of strychnine bound to GBP. A similar color scheme as shown in Fig. 3 ▸ is used, with C positions of strychnine in black and C positions of acetate and ethanediol (EDO) in green. (c) The binding of strychnine to the human GlyR-α3 homomer from PDB entry 5cfb (Huang et al., 2015 ▸); the residue numbers in the PDB entry are retained. (d) For comparative purposes the alignment of GBP [see Fig. 1 ▸(b)] with human GlyR-α3 is shown using the numbering scheme of the PDB entry. Residues shown in (c) are shown in gray for the principal side and in cyan for the complementary side.