PDBsum entry 4buo

Signaling protein

PDB id: 4buo

Contents

Protein chains

304 a.a.

Ligands

ARG-ARG-PRO-TYR-ILE-LEU ×2

GLY ×11

References listed in PDB file

Key reference

• Title: Structure of signaling-Competent neurotensin receptor 1 obtained by directed evolution in escherichia coli.
• Authors: P.Egloff, M.Hillenbrand, C.Klenk, A.Batyuk, P.Heine, S.Balada, K.M.Schlinkmann, D.J.Scott, M.Schütz, A.Plückthun.
• Ref.: Proc Natl Acad Sci U S A, 2014, 111, E655. [DOI no: 10.1073/pnas.1317903111]
• PubMed id: 24453215

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.