Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized ¹²⁹Xe NMR

Area of Science:

• Biophysics
• Structural Biology
• Neuroscience

Background:

• G protein-coupled receptors (GPCRs) exhibit diverse conformational states influencing downstream signaling.
• Characterizing these conformational dynamics is crucial for understanding receptor function.
• Cysteine labeling coupled with probes offers a method to detect receptor states.

Purpose of the Study:

• To introduce and validate the use of Nuclear Magnetic Resonance (NMR) of hyperpolarized 129Xe for detecting conformational states of the human neuropeptide Y2 receptor (NPY2R).
• To investigate the conformational landscape of NPY2R using a cryptophane-A cage attached to extracellular loop 2.
• To correlate spectroscopic signals with specific receptor structural states.

Main Methods:

• Site-specific cysteine introduction into the human NPY2R.
• Attachment of a xenon-trapping molecule, cryptophane-A, to the engineered cysteine.
• Application of chemical exchange saturation transfer (CEST) experiments using NMR of hyperpolarized 129Xe.
• Analysis of spectra in the absence (apo) and presence of the native ligand, neuropeptide Y (NPY).
• Molecular dynamics (MD) simulations to correlate spectral signals with structural conformations.

Main Results:

• High-quality 129Xe NMR spectra were obtained, reflecting distinct structural states of the NPY2R-cryptophane-A conjugate.
• Five distinct spectroscopic signals were assigned to the apo form of the receptor-cage conjugate.
• Upon addition of NPY, one new signal emerged, and existing signals showed subtle modifications, indicating ligand-induced conformational changes.
• MD simulations revealed frequent interactions between the xenon cage and the receptor, with a preference for interaction with the bound NPY ligand.

Conclusions:

• Hyperpolarized 129Xe NMR is a viable technique for probing the conformational states of GPCRs, exemplified by the NPY2R.
• The cryptophane-A probe attached to NPY2R allows for the detection of ligand-dependent structural rearrangements.
• This approach provides a powerful tool for quantifying the conformational landscape of receptors and their interactions with ligands.