PDBsum entry 4buo

Signaling protein

PDB id: 4buo

Contents

Protein chains

304 a.a.

Ligands

ARG-ARG-PRO-TYR-ILE-LEU ×2

GLY ×11

PDB id:

4buo

Name:

Signaling protein

Title:

High resolution structure of thermostable agonist-bound neurotensin receptor 1 mutant without lysozyme fusion

Structure:

Neurotensin receptor type 1. Chain: a, b. Fragment: residues 50-272 and 291-390. Synonym: nt-r-1, ntr1, high-affinity levocabastine-insensitive neurotensin receptor, ntrh. Engineered: yes. Mutation: yes. Other_details: thermostable mutant with intracellular loop 3 deletion b (e273-t290).

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Expressed in: escherichia coli. Expression_system_taxid: 469008. Expression_system_variant: tuner.

Resolution:

2.75Å R-factor: 0.249 R-free: 0.273

Authors:

P.Egloff,M.Hillenbrand,K.M.Schlinkmann,A.Batyuk,P.Mittl,A.Plueckthun

Key ref:

P.Egloff et al. (2014). Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc Natl Acad Sci U S A, 111, E655. PubMed id: 24453215 DOI: 10.1073/pnas.1317903111

Date:

21-Jun-13 Release date: 29-Jan-14

Protein chains

P20789  (NTR1_RAT) -  Neurotensin receptor type 1 from Rattus norvegicus

Seq: 424 a.a.

Struc: 304 a.a.*

* PDB and UniProt seqs differ at 11 residue positions (black crosses)

DOI no: 10.1073/pnas.1317903111

Proc Natl Acad Sci U S A 111:E655 (2014)

PubMed id: 24453215

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

P.Egloff, M.Hillenbrand, C.Klenk, A.Batyuk, P.Heine, S.Balada, K.M.Schlinkmann, D.J.Scott, M.Schütz, A.Plückthun.

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.