4BV0

High Resolution Structure of Evolved Agonist-bound Neurotensin Receptor 1 Mutant without Lysozyme Fusion

Summary for 4BV0

• Entry DOI: 10.2210/pdb4bv0/pdb
• Related: 3ZEV 4BV0 4BWB
• Descriptor: NEUROTENSIN RECEPTOR TYPE 1, NEUROTENSIN/NEUROMEDIN N (2 entities in total)
• Functional Keywords: signaling protein, g protein coupled receptor, membrane protein
• Biological source: RATTUS NORVEGICUS (NORWAY RAT); More
• Total number of polymer chains: 4
• Total formula weight: 77613.03

Authors

Egloff, P.,Hillenbrand, M.,Scott, D.J.,Schlinkmann, K.M.,Heine, P.,Balada, S.,Batyuk, A.,Mittl, P.,Schuetz, M.,Plueckthun, A. (deposition date: 2013-06-24, release date: 2014-01-29, Last modification date: 2024-11-13)

Primary citation

Egloff, P.,Hillenbrand, M.,Klenk, C.,Batyuk, A.,Heine, P.,Balada, S.,Schlinkmann, K.M.,Scott, D.J.,Schuetz, M.,Plueckthun, A.
Structure of Signaling-Competent Neurotensin Receptor 1 Obtained by Directed Evolution in Escherichia Coli
Proc.Natl.Acad.Sci.USA, 111:E655-E662, 2014

PubMed Abstract: 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.

PubMed: 24453215
DOI: 10.1073/PNAS.1317903111

Experimental method

X-RAY DIFFRACTION (3.1 Å)