1. Engineering a surrogate human heteromeric α/β glycine receptor orthosteric site exploiting the structural homology and stability of acetylcholine-binding protein
- Author
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Alice Dawson, Paul Trumper, Juliana Oliveira de Souza, Holly Parker, Mathew J. Jones, Tim G. Hales, and William N. Hunter
- Subjects
acetylcholine-binding protein ,crystal structures ,glycine receptor ,ligand-gated ion channel ,nicotine ,strychnine ,tropisetron ,Crystallography ,QD901-999 - 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.
- Published
- 2019
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