Complex of an Active μ-Opioid Receptor with a Cyclic Peptide Agonist Modeled from Experimental Constraints

Abstract

Site-directed mutagenesis and design of Zn(2+)-binding centers have been used to determine a set of specific tertiary interactions between the mu-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), and its cyclic peptide agonist ligand, Tyr(1)-c(S-Et-S)[d-Cys(2)-Phe(3)-d-Pen(4)]NH(2) (JOM6). The binding affinity of the tetrapeptide is strongly dependent on the nature of its first and third residues and on substitutions at positions 213, 216, 237, 300, 315, and 318 of the mu-opioid receptor. His(1) and His(3) analogues of the ligand were able to form metal-binding complexes with the V300C and G213C/T315C receptor mutants, respectively. Direct contact of the Phe(3) residue of JOM6 with Gly(213), Asp(216), Thr(315), and Trp(318) of the receptor was suggested by the binding affinities of His(3)-, Nle(3)-, Leu(3)-, Aci(3)-, Delta(E)Phe(3)-, and Delta(Z)Phe(3)-substituted peptides with the G213C/T315C, D216V, T315C, and W318L mutants. The improved binding affinity of the free carboxylate analogue of JOM6 for binding to the E229D mutant revealed an interaction between the C-terminal group of the peptide and Glu(229) of the receptor. The experimental constraints that were obtained were applied for distance geometry modeling of the mu-receptor in complex with the tetrapeptide agonist ligand, JOM6. The active conformation of the opioid receptor was calculated using the crystal structure of "inactive" rhodopsin and published engineered and intrinsic metal-binding sites and disulfide bonds that allow or facilitate activation of GPCRs. Interhelical H-bonds existing in the mu-receptor were applied as additional distance constraints. The calculated model of the receptor-ligand complex can serve as a prototype of the active state for all rhodopsin-like GPCRs. It displays a strongly shifted transmembrane helix 6 (TM6) and reorientation of the conserved Trp(293) residue in TM6 upon its interaction with the agonist. Importantly, the binding pockets of the active and inactive states are not identical, which implies distinct interaction modes of agonists and antagonists. In the active state, the binding pocket of the mu-receptor is complementary to the previously proposed receptor-bound conformation of JOM6.

Interactions of Human Melanocortin 4 Receptor with Nonpeptide and Peptide Agonists,

“…The experimental constraints can be determined using a variety of techniques (31,32). For example, the experimental studies of the effects of numerous mutations on binding of natural antagonists to hMC4R allowed modeling of the complex of the inactive conformation of hMC4R with AGRP and agouti protein (33).…”

“…The inactive state of hMC4R (SwissProt accession code P32245) has recently been modeled from the rhodopsin crystal structure (24) by iterative distance geometry refinement (33), an approach described and tested previously (29)(30)(31)40 An alterative conformation of EL3 was also used with TM6 extended by three residues at the extracellular end. Sequence alignment of MCRs and rhodopsin (Figure 2) has been described previously (33).…”

“…The dihedral angles of receptor residues were generally taken as in the template, with allowed deviations of 30°. Side chain rotamers were taken from the model of the inactive hMC4R (33), with the exception of several residues that rotate upon receptor activation (31) (41). The pairwise r.m.s.d.…”

“…For example, the experimental studies of the effects of numerous mutations on binding of natural antagonists to hMC4R allowed modeling of the complex of the inactive conformation of hMC4R with AGRP and agouti protein (33). Moreover, we have recently calculated the model of active conformation of the μ-opioid receptor, using distance constraints from the crystal structure of the inactive conformation of rhodopsin (24) together with a large set of experimental constraints that are compatible with active states in several GPCRs (31). This model of the active conformation of μ-receptor can be used as a structural template for the modeling of other rhodopsin-like GPCRs that apparently share common activation mechanism and similar active conformations (34,35).…”

“…More specifically, we have identified hMC4R residues required for activity of two small-molecule, peptidomimetic agonists, THIQ and MB243, by examining the effects of thirteen mutations in the TM domain on agonist binding and ligand-induced receptor-mediated cAMP accumulation. A homology model of the active hMC4R state was generated, based on our previous experimental and modeling studies of the inactive hMC4R (33) and of the μ-opioid receptor in the active state (31) and was used 3′, 5′-adenosine monophosphate (cAMP) measurement cAMP measurements were performed using a competitive binding assay kit (TRK 432, Amersham; Arlington Heights, IL) as previously described (38). 2 × 10 5 cells were planted on 24 well plates and cultured for ∼17-19 h before the experiments.…”