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

Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):E655-62. doi: 10.1073/pnas.1317903111. Epub 2014 Jan 22.


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.

Keywords: detergents; membrane proteins; protein engineering; protein stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Crystallography, X-Ray
  • Directed Molecular Evolution*
  • Escherichia coli / genetics*
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Stability
  • Receptors, Neurotensin / chemistry
  • Receptors, Neurotensin / genetics*
  • Sequence Homology, Amino Acid
  • Signal Transduction*


  • Receptors, Neurotensin

Associated data

  • PDB/3ZEV
  • PDB/4BUO
  • PDB/4BV0
  • PDB/4BWB