Abstract
Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS1). We establish that the NTS1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination.
| Original language | English (US) |
|---|---|
| Article number | 112015 |
| Journal | Cell reports |
| Volume | 42 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 31 2023 |
Funding
We would like to thank Dr. Piotr Mroz (Indiana University) for synthesis of NT8-13, Dr. Vanta Jameson and Dr. Josh Kie (The University of Melbourne) for assistance with flow cytometry, and Prof. Andrew L. Lee (The University of North Carolina at Chapel Hill) for fruitful discussions. The 14.1 T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. The project was funded by KAKENHI 21H04791 (A.I.) and 21H051130 (A.I.); JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science ( JSPS ); LEAP JP20gm0010004 (A.I.); BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development ( AMED ); FOREST JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency ( JST ); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigación, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovación , María de Maseztu grant CEX2021-001202-M (J.C.P. and M.P.); Australian National Health and Medical Research Council ( NHMRC ) grants 1081844 and 1141034 (R.A.D.B., D.J.S., and P.R.G.); Indiana Precision Health Initiative (J.J.Z.); and National Institutes of Medicine ( NIH ) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.). We would like to thank Dr. Piotr Mroz (Indiana University) for synthesis of NT8-13, Dr. Vanta Jameson and Dr. Josh Kie (The University of Melbourne) for assistance with flow cytometry, and Prof. Andrew L. Lee (The University of North Carolina at Chapel Hill) for fruitful discussions. The 14.1 T spectrometer at Indiana University used in this study was generously supported by the Indiana University Fund. The project was funded by KAKENHI 21H04791 (A.I.) and 21H051130 (A.I.); JPJSBP120213501 (A.I.) from Japan Society for the Promotion of Science (JSPS); LEAP JP20gm0010004 (A.I.); BINDS JP20am0101095 (A.I.) from the Japan Agency for Medical Research and Development (AMED); FOREST JPMJFR215T (A.I.) and JST Moonshot Research and Development Program JPMJMS2023 (A.I.) from Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science (A.I.); Takeda Science Foundation (A.I.); Ono Medical Research Foundation (A.I.); Uehara Memorial Foundation (A.I.); Agencia Estatal de Investigación, Spain grant PID2019-104914RB-I00 (J.C.P. and M.P.); Ministerio de Ciencia e Innovación, María de Maseztu grant CEX2021-001202-M (J.C.P. and M.P.); Australian National Health and Medical Research Council (NHMRC) grants 1081844 and 1141034 (R.A.D.B. D.J.S. and P.R.G.); Indiana Precision Health Initiative (J.J.Z.); and National Institutes of Medicine (NIH) grants R00GM115814 (J.J.Z.) and R35GM143054 (J.J.Z.). F.B. M.P. A.I. R.A.D.B. D.J.S. P.R.G. and J.J.Z. designed experiments; F.B. and F.Y. prepared NMR samples; F.B. M.P. A.I. J.C.P. and J.J.Z. performed experiments and analyzed data; F.B. and J.J.Z. wrote the manuscript; and all authors reviewed and edited the manuscript. The authors declare no competing financial interests. We support inclusive, diverse, and equitable conduct of research.
Keywords
- CP: Cell biology
- DFT
- G protein-coupled receptors
- GPCRs
- NMR
- allosteric modulator
- allostery
- biased agonist
- conformational entropy
- conformational selection
- density functional theory
- dynamics
- methionine
- pharmacological efficacy
ASJC Scopus subject areas
- General Biochemistry, Genetics and Molecular Biology