Refinement of a homology model of the mu-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) alpha 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 mu-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 Zn(2+) (IC(50) approximately 30microM), we observed that mutation of His(319) (TM7) abolished Zn(2+) inhibition of ligand binding, while mutation of Asp(216) (extracellular loop 2) decreased the effect of Zn(2+), suggesting these residues participate in the intrinsic Zn(2+)-binding center of MOR. To verify the relative orientation of TM5 and TM6 and to examine whether a rhodopsin-like alpha aneurism is present in TM5, we engineered Zn(2+)-binding centers by mutating residues of TM5 and TM6 to Cys or His, making use of the native His(297) in TM6 as an additional Zn(2+)-coordination site. Inhibition of opioid ligand binding by Zn(2+) suggests that residues Ile(234) and Phe(237) in TM5 face the binding-site crevice and form a metal-binding center with His(297) and Val(300) in TM6. This observation is inconsistent with a rhodopsin-like structure, which would locate Ile(234) on the lipid-exposed side of TM5, too distant from other residues making up the Zn(2+)-binding site. Subsequent distance geometry refinement of the MOR model indicates that the rhodopsin-like alpha aneurism is likely absent in TM2 but present in TM5.
Fowler
Carol B
CB
Department of Medicinal Chemistry, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065, USA.
Pogozheva
Irina D
ID
LeVine
Harry
H
Mosberg
Henry I
HI
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D000882:Haplorhini; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism*
D017450:Receptors, Opioid, mu; D001224:Aspartic Acid; D006639:Histidine; D015032:Zinc