Engineering a human P2X2 receptor with altered ligand selectivity in yeast

Elizabeth C Gardner; Caitlin Tramont; Petra Bachanová; Chad Wang; Hannah Do; Daniel R Boutz; Shaunak Kar; Boris V Zemelman; Jimmy D Gollihar; Andrew D Ellington

doi:10.1016/j.jbc.2024.107248

Engineering a human P2X2 receptor with altered ligand selectivity in yeast

J Biol Chem. 2024 Mar 29;300(5):107248. doi: 10.1016/j.jbc.2024.107248. Online ahead of print.

Authors

Affiliations

¹ Department of Molecular Biosciences, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas, USA.
² Antibody Discovery and Accelerated Protein Therapeutics, Department of Pathology & Genomic Medicine, Houston Methodist Research Institute, Houston, Texas, USA.
³ Department of Neuroscience, Center for Learning and Memory, The University of Texas at Austin, Austin, Texas, USA. Electronic address: zemelmanb@mail.clm.utexas.edu.
⁴ Antibody Discovery and Accelerated Protein Therapeutics, Department of Pathology & Genomic Medicine, Houston Methodist Research Institute, Houston, Texas, USA. Electronic address: jgollihar2@houstonmethodist.org.
⁵ Department of Molecular Biosciences, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas, USA. Electronic address: ellingtonlab@gmail.com.

Abstract

P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker's yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity toward the ATP analog AMP-PNP. The F303Y A304W variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304W P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.

Keywords: P2X2; directed evolution; genetic engineering; ligand-gated ion channel; synthetic biology; yeast humanization.