Structural Dynamics and Thermostabilization of Neurotensin Receptor 1

[Image: see text] The neurotensin receptor NTSR1 binds the peptide agonist neurotensin (NTS) and signals preferentially via the G(q) protein. Recently, Grisshammer and co-workers reported the crystal structure of a thermostable mutant NTSR1-GW5 with NTS bound. Understanding how the mutations thermostabilize the structure would allow efficient design of thermostable mutant GPCRs for protein purification, and subsequent biophysical studies. Using microsecond scale molecular dynamics simulations (4 μs) of the thermostable mutant NTSR1-GW5 and wild type NTSR1, we have elucidated the structural and energetic factors that affect the thermostability and dynamics of NTSR1. The thermostable mutant NTSR1-GW5 is found to be less flexible and less dynamic than the wild type NTSR1. The point mutations confer thermostability by improving the interhelical hydrogen bonds, hydrophobic packing, and receptor interactions with the lipid bilayer, especially in the intracellular regions. During MD, NTSR1-GW5 becomes more hydrated compared to wild type NTSR1, with tight hydrogen bonded water clusters within the transmembrane core of the receptor, thus providing evidence that water plays an important role in improving helical packing in the thermostable mutant. Our studies provide valuable insights into the stability and functioning of NTSR1 that will be useful in future design of thermostable mutants of other peptide GPCRs.