Dynamics of the Human Y1-Receptor in the Inactive and APO States Investigated by Solid-State Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulations

Abstract

The Y1 receptor (Y1R) is part of the G protein-coupled receptor (GPCR) superfamily, which plays a crucial role in many biological signaling processes and is of high interest for medical applications. It is involved in many physiological and pathophysiological functions, related to obesity and cancer. Y1R is activated by neuropeptide Y (NPY), which is involved in several physiological processes including the stimulation of food intake. Here, we studied the receptor dynamics bound to UR-MK299, which is a small molecular weight antagonist, and in the apo form both embedded in POPC membranes at 37 °C. For investigation by nuclear magnetic resonance (NMR) spectroscopy the receptor was labeled uniformly with 13C. This allowed investigating its dynamics by measurement of order parameters. However, due to the uniform labeling the NMR order parameters are averaged over the whole receptor without any site resolution and therefore could only show general differences between the different states. To obtain information of individual segmental motions, we conducted molecular dynamics (MD) simulations of both states using the recently published crystal structure in presence of UR-MK299 (PDB entry code: 5ZBQ). For the apo state, the antagonist was removed and the receptor relaxed for several microseconds before analysis. For the UR-MK299 bound state the CHARMM force field of the antagonist was parametrized with Gaussian. The simulations were prepared following a newly developed general scheme for setup of GPCR MD simulations. The production runs were conducted for several microseconds and compared to the experimentally available data followed by a detailed analysis of the segmental motions. Aim of this analysis is to elucidate the complex structure function dynamics relationship of GPCRs combining state-of-the-art biophysical methods to obtain their time resolved motions.