Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry

Abstract

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K_D of 138 ± 55 nM, a ΔH of −9708 ± 1195 cal/mol and a ΔS of −1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K_D of 3.2 ± 0.8 μM, a ΔH of −2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.

Keywords::

• Pentameric ligand gated ion channel
• construct design
• isothermal titration calorimetry
• Pichia pastoris

Acknowledgements

We acknowledge financial support from HEALTH-F4-2007-201924 (EDICT). We thank Ewa Nilsson and David Månsson for assistance with the generation of different P. pastoris constructs, Fritz Schweikart for N-terminal sequencing and Paul Wan for scientific discussions and proof-reading the manuscript.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.