期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：6
页码：E655-E662
DOI：10.1073/pnas.1317903111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 A 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.
关键词：membrane proteins ; protein stability ; protein engineering ; detergents
