Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli.
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.
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Pascal
P
Egloff
Matthias
M
Hillenbrand
Christoph
C
Klenk
Alexander
A
Batyuk
Philipp
P
Heine
Stefanie
S
Balada
Karola M
KM
Schlinkmann
Daniel J
DJ
Scott
Marco
M
Schütz
Andreas
A
Plückthun
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055550:Protein Stability; D018028:Receptors, Neurotensin / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D018028:Receptors, Neurotensin
detergents; membrane proteins; protein engineering; protein stability