Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-02, Vol.111 (6), p.E655-E662
• Main Authors: Egloff, Pascal, Hillenbrand, Matthias, Klenk, Christoph, Batyuk, Alexander, Heine, Philipp, Balada, Stefanie, Schlinkmann, Karola M, Scott, Daniel J, Schütz, Marco, Plückthun, Andreas
• Format: Article
• Language: English
• Subjects: agonists; Amino Acid Sequence; Amino acids; antagonists; Biological Sciences; Crystallization; crystallography; Crystallography, X-Ray; Diffusion; directed evolution; Directed Molecular Evolution; E coli; Escherichia coli; Escherichia coli - genetics; G-protein coupled receptors; Models, Molecular; Molecular Sequence Data; Neuropeptides; palmitoylation; Pharmacology; PNAS Plus; Protein Stability; Receptors, Neurotensin - chemistry; Receptors, Neurotensin - genetics; Sequence Homology, Amino Acid; Signal Transduction; vapors; X-radiation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1317903111

Crystallography has advanced our understanding of G protein–coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled receptor signaling, receptor maturation, and desensitization.