Characterising the conformational diversity of the neurotensin receptor 1

© 2016 Dr. Fabian BumbakG Protein-Coupled Receptors (GPCRs) deliver approximately 80% of all signals across the cell membrane and are targets for around 26% of all prescription drugs. Conversely, over 85% of the GPCR family remain undrugged, often despite clear linkage to disease. Important reasons for this are that high-throughput screening has failed to deliver optimal drug leads and a lack of structural knowledge about how ligands engage GPCRs has hindered compound optimisation. The neurotensin receptor 1 (NTS1) is a GPCR for the tridecapeptide neurotensin (NT). Activation of NTS1 by NT has been implicated in schizophrenia, Parkinson’s disease and hypothermia, as well as cancer progression and drug abuse. Crystal structures of NTS1 bound to fragments of NT, as well as mutagenesis data, have defined intermediate states of NTS1 along with the high affinity binding site for NT. However, little is known about this receptor’s conformational dynamics and conformations that mediate particular signalling responses upon ligand interaction. Furthermore, transient interactions that may underlie NT binding remain uncharted. Like most integral membrane proteins, GPCRs are unstable in isolation, while recombinant expression yields remain poor. To overcome those hurdles, our laboratory used cellular high-throughput encapsulation, solubilisation and screening (CHESS)-based directed evolution to generate thermostable mutants of NTS1 suitable for biophysical studies. This thesis outlines expression of CHESS-derived NTS1 mutants to the E. coli inner membrane, their reconstitution in n-dodecyl β-D-maltopyranoside (DDM) micelles and their purification close to heterogeneity in the absence of stabilising ligands. Using small angle X-ray scattering (SAXS) we could show that our NTS1 mutant is monomeric when reconstituted in DDM micelles while a label free cell signalling assay indicated that it is signalling competent despite being thermostabilised. Dissociation constants (Kd’s) for a number of NT truncates as well as non-peptide ligands could be determined for one of our NTS1 mutants by means of surface plasmon resonance (SPR) spectroscopy. Saturation transfer difference NMR (STD NMR) was applied to detect low affinity peptide interactions to our engineered NTS1 mutants. The high stability of these receptors in detergent allowed us to repeatedly measure the binding of NT fragments over periods of up to 96 h at 25°C without significant loss of STD binding signal. Analysis of STD spectra enabled epitope mapping of the NT10-13/NTS1 interface. Molecular dynamics (MD) simulations of both NT8-13 and NT10-13 in complex with a homology model of our NTS1 mutant supported our solution NMR derived NT binding pose, which is not in complete agreement with available crystal structures. We show that the Y11 sidechain of NT10-13 is likely to populate two distinct conformations while the crystal structures only reveal one conformation. Furthermore, utilising a methionine biosynthesis pathway inhibition strategy we were able to achieve more than 95% 13CH3-methionine labelling of our NTS1 mutant in E. coli. Using 1H-13C-HMQC NMR, differential chemical shift and line-width changes of 13CH3-methionine resonances were observed upon binding of a full agonist, a β-arrestin biased agonist and a high-affinity antagonist to the 13CH3-methionine labelled receptor. These differences indicated that the NTS1 mutant exhibits conformational heterogeneity despite being thermostabilised with mutations. Assignment of the 10 13CH3-methionine resonances present in our receptor is being pursued using mutagenesis. Stably expressing minimal methionine mutants were generated by in vitro ‘sexual’ evolution through the PCR-based staggered extension process (StEP) and E. coli cells expressing the mutants were selected using fluorescence activated cell sorting (FACS) for their ability to bind fluorescently labelled agonist. Selected methionines will act as local probes in NMR based dynamics studies to further our understanding of the conformational responses of NTS1 upon ligand interaction