High Affinity Agonistic Metal Ion Binding Sites within the Melanocortin 4 Receptor Illustrate Conformational Change of Transmembrane Region 3

Abstract

We created a molecular model of the human melanocortin 4 receptor (MC4R) and introduced a series of His residues into the receptor protein to form metal ion binding sites. We were able to insert micromolar affinity binding sites for zinc between transmembrane region (TM) 2 and TM3 where the metal ion alone was able to activate this peptide binding G-protein-coupled receptor. The exact conformation of the metal ion interactions allowed us to predict the orientation of the helices, and remodeling of the receptor protein indicated that Glu 100 and Ile 104 in TM2 and Asp 122 and Ile 125 in TM3 are directed toward a putative area of activation of the receptor. The molecular model suggests that a rotation of TM3 may be important for activation of the MC4R. Previous models of G-protein-coupled receptors have suggested that unlocking of a stabilizing interaction between the DRY motif, in the cytosolic part of TM3, and TM6 is important for the activation process. We suggest that this unlocking process may be facilitated through creation of a new interaction between TM3 and TM2 in the MC4R.The G-protein-coupled receptors (GPCRs) 1 require a membrane to maintain their functionality and structural integrity. This makes crystallization of these receptors difficult, hampering structural determination. Crystallization of the first mammalian GPCR, the bovine rhodopsin, was an important breakthrough (1). Earlier three-dimensional models of GPCRs were mainly based on cryoelectron microscopy data generated from bacteriorhodopsin (2), assuming a common fold in the transmembrane (TM) regions. Such assumptions had clear limitations as bacteriorhodopsin is not a GPCR and has no sequence homology to human GPCRs. Even though the bovine rhodopsin model is detailed, it is not clear how applicable it is for different GPCRs, considering the large variety of these receptors in the human genome. Today it is still an unrealistic task to crystallize the hundreds of GPCRs found in the human genome mainly due to the difficulties in obtaining material suitable for solubilization and crystallization studies. Site-directed mutagenesis has played an important role in determining the putative interaction of a ligand to a single amino acid within a GPCR. Such interactions, however, may not always be informative about the orientation of the helix bundles, which is crucial information for building structural models. Moreover, the results of alanine replacement studies in most cases cannot discriminate between specific ligand-receptor interactions and changes that cause unspecific conformational alterations that perturb the binding. This is particularly evident when the ligand is a flexible molecule like a peptide or a protein. One alternative approach for studying the three-dimensional structure of GPCRs is construction of a high affinity zinc-binding site between the helices, using two His residues facing each other (3). Such artificial intrahelical and interhelical binding sites have been used effectively to determine the orientation and exact distance...

Pharmacological Characterization of Loss of Function Mutations of the Human Melanocortin 1 Receptor That Are Associated with Red Hair

“…The binding method has been very reliable and reproducible for a long period in the lab independent of the cell type used. We have compared the two cell types, COS (CV Origin SV 40) and HEK293-EBNA, several times on our lab, and found that they give the same results in binding studies (Ringholm et al, 2002;Lagerströ m et al, 2003). The Val92Met mutant had, however, clearly lower affinity to a-MSH (about 100-fold) comparing to the wild-type receptor.…”

Receptor−Antagonist Interactions in the Complexes of Agouti and Agouti-Related Protein with Human Melanocortin 1 and 4 Receptors,

“…To improve the accuracy of the modeling and to avoid the common modeling errors related to the structural misalignment and erroneous loop modeling (30,49), we used model refinement by satisfying structural restraints (31). The restraints were derived not only from the structural templates (1gzm for the receptor and 1hyk and 1mr0 for the ligand) but also from mutagenesis experiments demonstrating the formation of Zn 2+ -binding sites in MCRs (39)(40)(41)44). The native and non-natural disulfide bonds, which can be formed in wildtype or mutant receptors, provide the additional structural restrains.…”

Modeling activated states of GPCRs: the rhodopsin template

“…We use only the C b -C b constraint because the data were obtained either for GPCRs other than rhodopsin [52][53][54][55] or for engineered sites in rhodopsin [56]. We use C b -C b distance >8.5 Å if the Zinc binding site blocks activation and 6.5…”