Refinement of a Homology Model of the μ-Opioid Receptor Using Distance Constraints from Intrinsic and Engineered Zinc-Binding Sites

Publication of the rhodopsin X-ray structure has facilitated the development of homology models of other G protein-coupled receptors. However, possible shifts of transmembrane (TM) α helices, expected variations in helical distortions, and differences in loop size necessitate experimental verificati...

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Bibliographic Details
Published in:Biochemistry (Easton) 2004-07, Vol.43 (27), p.8700-8710
Main Authors: Fowler, Carol B, Pogozheva, Irina D, LeVine, Harry, Mosberg, Henry I
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Aspartic Acid - metabolism
Binding Sites
COS Cells
Haplorhini
Histidine - metabolism
Humans
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Structure, Tertiary
Rats
Receptors, Opioid, mu - chemistry
Receptors, Opioid, mu - genetics
Receptors, Opioid, mu - metabolism
Sequence Alignment
Zinc - metabolism
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Summary:Publication of the rhodopsin X-ray structure has facilitated the development of homology models of other G protein-coupled receptors. However, possible shifts of transmembrane (TM) α helices, expected variations in helical distortions, and differences in loop size necessitate experimental verification of these comparative models. To refine a rhodopsin-based homology model of the μ-opioid receptor (MOR), we experimentally determined structural-distance constraints from intrinsic and engineered metal-binding sites in the rat MOR. Investigating the relatively high intrinsic affinity of MOR for Zn2+ (IC50 ∼ 30μM), we observed that mutation of His319 (TM7) abolished Zn2+ inhibition of ligand binding, while mutation of Asp216 (extracellular loop 2) decreased the effect of Zn2+, suggesting these residues participate in the intrinsic Zn2+-binding center of MOR. To verify the relative orientation of TM5 and TM6 and to examine whether a rhodopsin-like α aneurism is present in TM5, we engineered Zn2+-binding centers by mutating residues of TM5 and TM6 to Cys or His, making use of the native His297 in TM6 as an additional Zn2+-coordination site. Inhibition of opioid ligand binding by Zn2+ suggests that residues Ile234 and Phe237 in TM5 face the binding-site crevice and form a metal-binding center with His297 and Val300 in TM6. This observation is inconsistent with a rhodopsin-like structure, which would locate Ile234 on the lipid-exposed side of TM5, too distant from other residues making up the Zn2+-binding site. Subsequent distance geometry refinement of the MOR model indicates that the rhodopsin-like α aneurism is likely absent in TM2 but present in TM5.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi036067r