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

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Abstract
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.