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• W2010292831 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 Glu100 and Ile104 in TM2 and Asp122 and Ile125 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. 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 Glu100 and Ile104 in TM2 and Asp122 and Ile125 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) 1The abbreviations used are: GPCRG-protein-coupled receptorMC4Rmelanocortin 4 receptorTMtransmembrane. 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 break-through (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). Earlier three-dimensional models of GPCRs were mainly based on cryoelectron microscopy data generated from bacteriorhodopsin (2Schertler G.F. Villa C. Henderson R. Nature. 1993; 362: 770-772Crossref PubMed Scopus (714) Google Scholar), 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 (3Elling C.E. Thirstrup K. Nielsen S.M. Hjorth S.A. Schwartz T.W. Ann. N. Y. Acad. Sci. 1997; 814: 142-151Crossref PubMed Scopus (22) Google Scholar). Such artificial intrahelical and interhelical binding sites have been used effectively to determine the orientation and exact distances between the α-helices of the tachykinin, opioid, and the β-adrenergic receptor families (4Thirstrup K. Elling C.E. Hjorth S.A. Schwartz T.W. J. Biol. Chem. 1996; 271: 7875-7878Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 5Elling C.E. Schwartz T.W. EMBO J. 1996; 1: 6213-6219Crossref Scopus (87) Google Scholar, 6Elling C.E. Thirstrup K. Holst B. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12322-12327Crossref PubMed Scopus (104) Google Scholar). Moreover, in two previous studies (6Elling C.E. Thirstrup K. Holst B. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12322-12327Crossref PubMed Scopus (104) Google Scholar, 7Holst B. Elling C.E. Schwartz T.W. Mol. Pharmacol. 2000; 58: 263-270Crossref PubMed Scopus (56) Google Scholar), an interhelical binding site has been created that allowed the metal ion to act as an agonist and activate a GPCR. The coordination of the metal ion binding sites is well characterized in numerous x-ray structures of soluble proteins, and the distances between the chelating atoms and the metal ion are known, providing excellent specific information regarding the orientation of the relative helices (3Elling C.E. Thirstrup K. Nielsen S.M. Hjorth S.A. Schwartz T.W. Ann. N. Y. Acad. Sci. 1997; 814: 142-151Crossref PubMed Scopus (22) Google Scholar, 8Christianson D.W. Adv. Protein Chem. 1991; 42: 281-355Crossref PubMed Google Scholar). G-protein-coupled receptor melanocortin 4 receptor transmembrane. The MC4R is a Gs-coupled receptor belonging to the group of rhodopsin-like class α receptors (9Fredriksson R. Lagerstrom M.C. Lundin L.G. Schioth H.B. Mol. Pharmacol. 2003; 63: 1256-1272Crossref PubMed Scopus (2201) Google Scholar). The MCRs seem to be remarkably well conserved through evolution (10Ringholm A. Fredriksson R. Poliakova N. Yan Y.-L. Postlethwait J.H. Larhammar D. Schiöth H.B. J. Neurochem. 2002; 82: 6-18Crossref PubMed Scopus (104) Google Scholar). The MC4R is exclusively expressed in brain, particularly in hypothalamus, and has an important role for central regulation of food intake and energy balance. Agonistic stimulation of the receptor reduces food intake, whereas antagonists are among the most potent orexigenic agents available (11Kask A. Mutulis F. Muceniece R. Pahkla R. Mutule I. Wikberg J.E. Rago L. Schioth H.B. Endocrinology. 1998; 139: 5006-5014Crossref PubMed Scopus (148) Google Scholar). This receptor, and the closely related MC3R, also has an important role in regulating the metabolic rate and general body weight homeostasis. The MC4R is thus one of the most pursued targets for the development of drugs to treat obesity and anorexia (12Schioth H.B. Watanobe H. Brain Res. Brain Res. Rev. 2002; 38: 340-350Crossref PubMed Scopus (65) Google Scholar). In this study we constructed a molecular model of the human MC4R based on the crystal structure of rhodopsin (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). We also inserted His residues into several positions in the receptor in order to generate metal ion binding sites. We were able to create artificial high affinity zinc sites where the zinc ion was able to concentration dependently increase cAMP levels. The results were used to construct a refined model for the MC4R protein that suggests that interaction between TM2 and TM3 is important for the activation process of this receptor. Molecular Modeling—The model was based on the coordinates from the high resolution structure of bovine rhodopsin (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). The TM regions were reconstructed to avoid problems with structural features derived from specific amino acid sequences found in the rhodopsin receptor but not in the MC4R or vice versa. The resulting TM (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar, 2Schertler G.F. Villa C. Henderson R. Nature. 1993; 362: 770-772Crossref PubMed Scopus (714) Google Scholar, 3Elling C.E. Thirstrup K. Nielsen S.M. Hjorth S.A. Schwartz T.W. Ann. N. Y. Acad. Sci. 1997; 814: 142-151Crossref PubMed Scopus (22) Google Scholar, 4Thirstrup K. Elling C.E. Hjorth S.A. Schwartz T.W. J. Biol. Chem. 1996; 271: 7875-7878Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 5Elling C.E. Schwartz T.W. EMBO J. 1996; 1: 6213-6219Crossref Scopus (87) Google Scholar, 6Elling C.E. Thirstrup K. Holst B. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12322-12327Crossref PubMed Scopus (104) Google Scholar, 7Holst B. Elling C.E. Schwartz T.W. Mol. Pharmacol. 2000; 58: 263-270Crossref PubMed Scopus (56) Google Scholar) boundaries were between positions Gln43 and Ile69, Phe81 and Ile104, Ile121 and Ile151, Val163 and Tyr187, Ser190 and Met215, Asn240 and Leu265, and Ser282 and Tyr320 (including the postulated eight α-helix), respectively. The positions of the three first amino acids in the cytosolic part of the helices in the rhodopsin model were used to orientate the helical segments in relation to each other. Segments of the extra- and intracellular parts of the receptor were excluded from the model due to length differences between the rhodopsin and the MC4R and due to inaccuracy in computer predictions of random coil structures. Modeling was performed with Sybyl 6.4 software (Tripos, Germany) running on work station O2 (Silicon Graphics, Mountain View, CA). Helices were given ideal α-helical conformation, and charges were assigned and helical structure refined using energy minimization in the subroutine Powell. Minimizations were carried out until convergence at 0.05 kcal/(mol A) energy gradient difference between successive minimizations steps was reached (maximum iteration 1000, non-bonded cut-off = 8 Å, dielectric constant = 1). Initial optimization was carried out using simplex to decrease the number of iterations required for convergence. The whole model was then minimized giving a receptor model with the energy of –288.3 kcal/mol. Refinements of the model were made after mutational data and then re-minimized giving a final energy of –294.6 kcal/mol. Distance measurements were made between the α-carbons of the pinpointed residues. Construction of Receptor Mutants—Point mutations were introduced in the human MC4R by PCR in two steps using Pfx polymerase (Invitrogen). Specific end primers containing HindIII (Amersham Biosciences) and XhoI (Amersham Biosciences) sites were used together with mutated internal primers in two steps using the following conditions: 30 s at 95 °C for one cycle, then 30 s at 95 °C, 40 s at 50 °C and 1 min at 72 °C for 40 cycles followed by 5 min at 72 °C. In the first step, overlapping fragments containing the desired mutation were generated. Fragments were purified using Qiagen Gel Extraction MiniElute kit (Qiagen, Stockholm, Sweden) and subsequently used as templates for a second PCR amplification to obtain the full-length mutated receptor. These fragments were digested with HindIII and XhoI, purified with QIAquick PCR purification kit (Qiagen), and ligated into the modified pCEP4 turbo expression vector. The integrity of each construct was controlled with restriction analysis and DNA sequencing using ABI PRISM Dye Terminator cycle sequencing kit version 2.0 (Applied Biosystems) according to the manufacturer's recommendations. The extension products were analyzed on an ABI PRISM-310 Automated Sequencer (Applied Biosystems). Sequences were analyzed using Sequencer 3.0 (Gene Codes) and found to be identical to the sequence of the desired MC4R mutant. Expression of Mutant Receptor in HEK 293-EBNA—Semi-stable cell lines expressing the different constructs were made from HEK 293-EBNA cells through transfection with 15 μg of plasmid-DNA using FuGENE™ Transfection reagent (Roche Applied Science) was diluted in Opti-MEM medium (Invitrogen) according to the manufacturer's recommendations. After transfection, cells were grown in Dulbecco's modified Eagle's medium/Nut Mix F-12 without l-glutamine (Invitrogen) supplied with 10% fetal calf serum (Invitrogen), 2.4 mml-glutamine (Invitrogen), and 250 μg/ml gentamicin (Invitrogen), penicillin/streptomycin (100 units of penicillin, 100 μg of streptomycin/ml) (Invitrogen) for 48 h before addition of 200 μg/ml hygromycin B (Invitrogen). The cells were harvested in 1× PBS after 3 weeks of selection. Membrane Preparation—After the harvest, the cells were homogenized using an UltraTurrax. The cell suspension was centrifuged for 3 min at 1300 rpm, and the supernatant was recentrifuged for 15 min at 15,000 rpm. The cell pellet was resuspended in binding buffer containing 50 mm Tris-HCl, pH 7.4, 2.5 mm MgCl2, 1 mm CaCl2, and 150 mm NaCl. Receptor Binding Assays—Binding studies were performed using a buffer containing 50 mm Tris-HCl, pH 7.4, 2.5 mm MgCl2, 1 mm CaCl2, 150 mm NaCl (for optimization of buffer system see Supplemental Material, 1A–C), in a final volume of 100 μl for 3 h at room temperature using 10–20 μg of membrane preparation per well, aiming at 60–70% specific binding. Competition studies were performed with various concentrations of ZnCl2 (Sigma) or non-labeled NDP-MSH peptide (Neosystem, France) included in the incubation mixture along with 0.6 nm125I-labeled NDP-MSH. Nonspecific binding was defined as the amount of radioactivity remaining bound to the cell homogenate after incubation in the presence of 1000 nm unlabeled NDP-MSH. Incubation was terminated by filtration through GF/C filters, Filtermat A (Wallac Oy, Turku, Finland), which had been pre-soaked in 0.3% polyethyleneimine (Sigma), using a TOMTEC Mach III cell harvester (Orange, CT). The filters were washed with 5.0 ml of 50 mm Tris-HCl, pH 7.4, per well at 4 °C and dried at 60 °C. The dried filters were then treated with MeltiLex A (PerkinElmer Life Sciences) melt-on scintillator sheets and counted with Wallac 1450 (Wizard automatic Microbeta counter). The results were analyzed with Prism 3.0 GraphPad (San Diego, CA). All binding assays were performed in duplicate and repeated at least three times. Non-transfected HEK 293-EBNA cells did not show any specific binding for 125I-labeled NDP-MSH. The radioligand NDP-MSH was labeled by the chloramine-T method and purified by high performance liquid chromatography. Protein concentrations were measured using a Bio-Rad Protein Assay (Bio-Rad). cAMP Assay—cAMP was assayed on semi-stable HEK 293-EBNA cells pre-treated for 15 min at 37 °C with 250 μm isobutylmethylxanthine (Sigma). For antagonist studies, 10–20 μg of cells were incubated in a 300-μl reaction volume with ZnCl2 at three different concentrations together with various concentrations (serial dilutions) of NDP-MSH for 1 h at 37 °C. All points were done in duplicate, and the measurements for each concentration were repeated at least three times. Agonistic studies were performed with various concentrations (serial dilutions) of NDP-MSH or ZnCl2. Non-transfected HEK 293-EBNA cells showed no cAMP response to NDP-MSH or ZnCl2. The incubations were terminated by addition of 25 μl of 4.4 m perchloric acid and neutralized by addition of 45 μl of 5 m KOH. Levels of cAMP were then measured using radioassay (Amersham Biosciences). Molecular Modeling and Placement of Initial Mutations—A preliminary molecular model of the MC4R was generated using the coordinates from the high resolution structure of the inactive conformation of bovine rhodopsin (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). We deliberately did not take into account any constraint from previous mutagenesis studies on the MCRs (13Yang Y.K. Fong T.M. Dickinson C.J. Mao C. Li J.Y. Tota M.R. Mosley R. Van Der Ploeg L.H. Gantz I. Biochemistry. 2000; 39: 14900-14911Crossref PubMed Scopus (164) Google Scholar, 14Yang Y. Chen M. Lai Y. Gantz I. Georgeson K.E. Harmon C.M. J. Biol. Chem. 2002; 277: 20328-20335Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 15Haskell-Luevano C. Cone R.D. Monck E.K. Wan Y.P. Biochemistry. 2001; 40: 164-179Crossref Scopus (137) Google Scholar). Based on our preliminary model, His residues were inserted into several positions close to the extracellular parts of the receptor. These positions were predicted to be facing the binding pocket, thus enabling the formation of a zinc-binding site possible to induce activation of the receptor or to inhibit ligand binding thereon. In this way we created the following single mutants (among a number of others): MC4R-I103H and MC4R-I104H at the top of TM2, MC4R-I125H at the top of TM3, and MC4R-F284H and MC4R-N285H at the top of TM7. In the preliminary model Ile104 was orientated toward TM3, 11 Å away from the TM3 backbone. Position Ile103 was in close proximity to Ile104 but turned toward the lipid bilayer. Ile125 was facing TM2, 14 Å from the α-carbon of Ile104 but one helical turn up. Position Phe284 was placed in TM7, facing TM3 at a distance of 17 Å from the α-carbon of Ile125. Asn285 was positioned in TM7, 18 Å away from the backbone of Ile104 in TM2. Naturally occurring zinc-binding residues, His and Asp, in these areas of the TM regions were also investigated by creating Ala-substituted mutants: MC4R-D122A (TM3), MC4R-D126A (TM3), MC4R-H283A (TM7), and MC4R-D298A (TM7). Receptor Binding Analysis of Initial Mutants—At first we optimized the ligand binding conditions (see Supplemental Material, 1C). The binding results show that the wild-type MC4R bound zinc with about 20 μm affinity (Table I). This natural binding site for zinc in the human MC4R was reported during our studies by another group (16Holst B. Elling C.E. Schwartz T.W. J. Biol. Chem. 2002; 277: 47662-47670Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These results are in agreement with ours. The introduction of the His residues listed above did not affect the binding of NDP-MSH significantly. However, MC4R-I104H (TM2) and MC4R-I125H (TM3) both gained 26-fold in affinity for zinc compared with the wild-type receptor (Table I). Moreover, the MC4R-I103H mutant in TM2 showed a 49-fold increase in zinc affinity. We also found an increase in affinity to zinc for two mutants in TM7, MC4R-F284H and MC4R-N285H, 8- and 19-fold, respectively. As mentioned above several alanine mutations were made on residues that may have the capability to create a high affinity binding site for zinc. Several of these mutants showed reduced zinc binding affinity without greatly influencing the NDP-MSH binding. The MC4R-D122A (TM3) lost all detectable affinity to zinc but maintained its affinity to NDP-MSH. The MC4R-H283 (TM7) mutant showed a 21-fold decrease in affinity for zinc without affecting the binding to NDP-MSH. Replacing Asp in position 298, creating MC4R-D298A, did not affect the Ki value for NDP-MSH, although the affinity of zinc was reduced below 1000 nm or at least 500-fold. The MC4R-D126A (TM3) mutant did, in agreement with previous results on the mouse MC4R (14Yang Y. Chen M. Lai Y. Gantz I. Georgeson K.E. Harmon C.M. J. Biol. Chem. 2002; 277: 20328-20335Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), lose all affinity to NDP-MSH and was therefore impossible to study further.Table ICompetition binding experiments with NDP-MSH or ZnCl2 on HEK 293-EBNA cells expressing the human wild-type receptor (hMC4 wt) or MC4 mutant receptorReceptorKi NDP-MSHKi ZnCl2Fold change in ZnCl2 affinity (+/-)nmμmhMC4 wt12.3 ± 1.620.07 ± 1.33I103H58.0 ± 3.20.41 ± 0.07+49.0I104H6.73 ± 1.10.76 ± 0.03+26.4D122A37.2 ± 1.3>1000I125H39.4 ± 2.40.76 ± 0.04+26.4H126ANBaNB, no detectable binding.NBH283A9.43 ± 0.8429 ± 45-21.5F284H5.9 ± 0.612.52 ± 0.52+8.0N285H4.4 ± 0.61.07 ± 0.61+18.8D298A3.9 ± 0.7>1000a NB, no detectable binding. Open table in a new tab Studies of Intracellular Coupling and Further Mutagenesis— Repeated second messenger studies on the wild-type receptor revealed no increased levels of intracellular cAMP upon exposure to zinc in our assay. Our antagonistic studies unveiled an inhibitory effect of zinc at 32.9 μm (see Table II and Fig. 1) in accordance with the binding studies (Table I). In agreement with several previous studies, NDP-MSH stimulation showed an agonistic response of 8.9 nm for the wild-type construct. The single mutants mentioned above responded agonistically to NDP-MSH displaying decreased EC50 values in comparison with the wild-type receptor (Table III). Cells expressing these constructs showed no increase in basal cAMP production. Interestingly, zinc induced cAMP production with a potency of 5.9 μm (Table III) in cells expressing the MC4R-I104H mutant, whereas cells transfected with MC4R-I103H showed no agonistic response. Moreover, zinc was able to inhibit the agonistic response of NDP-MSH, in cells expressing the MC4R-I103H mutant, with a Ki value of 42.1 μm (Table II). We subsequently investigated whether zinc was forming an interhelical or intrahelical agonistic binding site between the His in position 104 and a naturally occurring amino acid residue in TM3 or TM2. The most likely residues according to the original model were Asp122 in TM3 and Glu100 in TM2. Therefore, we replaced these residues with Ala forming the double mutants MC4R-I104H/D122A and MC4R-I104H/E100A. Double mutant MC4R-I104H/D122A was unable to respond to zinc, whereas the double mutant MC4R-I104H/E100A showed an agonistic response to zinc at 0.7 μm. These data are shown in Fig. 2A.Table IIZinc induced inhibition of cAMP production on HEK 293-EBNA cells expressing the human wild type receptor (hMC4 wt) or the mutated receptor constructs MC4-I103H or MC4-D122AReceptorEC50 NDP-MSHIC50 ZnCl2nmμmhMC4 wt8.9 ± 0.4532.9I103H258 ± 24.942.1D122A115 ± 29.6110.0 Open table in a new tab Table IIIcAMP production in response to NDP-MSH or ZnCl2 in HEK 293-EBNA cells expressing the wild-type receptor (hMC4 wt) or mutated receptor constructsReceptorEC50 NDP-MSHEC50 ZnCl2nmμmhMC4 wt8.90 ± 0.45NSaNS, no stimulation.E100A905 ± 87NSI103H258 ± 25NSI104H43.0 ± 19.25.91 ± 0.92D122A115 ± 30NSI125H28.4 ± 1.43.87 ± 1.69I104H/D122A8960 ± 200NSI104H/I125H7.45 ± 4.513.00 ± 1.49I104H/E100A433 ± 2380.70 ± 0.34I125H/E100A3890 ± 2140NSI125H/D122A554 ± 622.18 ± 1.18a NS, no stimulation. Open table in a new tab Fig. 2A, generation of cAMP in response to Zn2+ for receptor mutant MC4R-I104H (▪), MC4R-I104H/E100A (♦), and MC4R-I104H/D122A (▴). Each point represents the average ± S.E. of values in duplicate. B, generation of cAMP in response to Zn2+ for receptor mutant MC4R-I125H (▪), MC4R-I125H/E100A (▴), and MC4R-I125H/D122A (♦). Each point represents the average ± S.E. of values in duplicate. Non-transfected HEK 293-EBNA cells showed no cAMP response to Zn2+ (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Investigations of the single His-substituted MC4R-I125H construct showed an agonistic response of 3.9 μm upon exposure to zinc. According to the original model, one highly plausible residue for interhelical interaction with the His in position 125 was Glu100 in TM2. There was also a possibility for an intrahelical interaction between I125H and Asp122. However, this did not appear to be the case since the double mutant MC4R-I125H/D122A responded to zinc with a potency of 2.2 μm. Upon replacement of Glu100 with Ala, the double mutant MC4R-E100A/I125H was unable to respond to zinc. These results are shown in Fig. 2B. Remodeling of the MC4R—An interaction between the carboxylate group of Asp or Glu and a zinc ion typically occurs when the metal-oxygen distance is in the range of 2.3–2.6 Å (8Christianson D.W. Adv. Protein Chem. 1991; 42: 281-355Crossref PubMed Google Scholar). Furthermore, the distance between the distant nitrogen in the imidazole of His and the zinc ion is expected to vary between 1.9 and 2.2 Å (17Chakrabarti P. Protein Eng. 1990; 4: 49-56Crossref PubMed Scopus (83) Google Scholar). After inclusion of the zinc atomic radius of 0.74 Å, a measurement between the respective α-carbons will increase the distance with 7.5–9.2 Å, giving a maximum distance of 12.3–14.8 Å between two zinc-chelating amino acids. Our preliminary MC4R model was re-defined in order to fit the data mentioned above. Accordingly, TM3 was rotated around its own longitudinal axis ∼76° counterclockwise, making the interacting residues in TM3 facing the corresponding residues in TM2 (Fig. 3, A and B). The distance between the α-carbon of Asp122 in TM3 and I104H in TM2 was calculated to 10 Å. The corresponding distance between the α-carbon of Glu100 in TM2 and I125H in TM3 was estimated to be 8 Å, both interactions clearly within the maximum chelating distance of 12.3–14.8 Å. The distances in the perimeter of the helical bundle were measured as described in Fig. 3D. Comparison between orientations in the crystallized bovine rhodopsin receptor and the remodeled human MC4R shows a clear spatial agreement (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar) (Fig. 3C). TM1 and TM2 show agreement regarding the orientations of the highly conserved residues Asn62 (TM1) and Asp90 (TM2). The rhodopsin model and our new model also share the same spatial arrangement for Asp298 and Tyr302 in the well conserved (D/N)PXXY motif in the bottom of TM7. The highly conserved Asp90 in TM2, which corresponds to Asp83 in bovine rhodopsin, is pointing toward the cavity between TM7, TM1, TM2, and TM3 as in the rhodopsin model, where it is participating in an interaction with Asp298 in TM7 (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar), and Trp258 in the well conserved CWXP motif in TM6 is directed toward the binding cavity. This amino acid is believed to interact with the C13-methyl group of retinal in the bovine rhodopsin model (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). Initially, we created a preliminary model of the MC4R based on the crystal structure of the bovine rhodopsin receptor. Subsequently, His residues were inserted into several positions in order to create artificial metal ion binding sites. The most interesting mutagenesis results were obtained from those at the extracellular parts of TM2 and TM3. Three of our mutants, MC4R-I103H (TM2), MC4R-I104H (TM2), and MC4R-I125H (TM3), remarkably increased the zinc affinity of the receptor by 25–50-fold. Moreover, zinc was able to induce cAMP production with a potency of 5.9 μm in cells expressing the MC4R-I104H mutant, whereas cells transfected with MC4R-I103H showed no agonistic response. Furthermore, zinc was able to inhibit the agonistic response of NDP-MSH in cells expressing the MC4R-I103H mutant, with a Ki value of 42.1 μm (Fig. 1). Interestingly, the single His-substituted MC4R-I125H construct also showed an agonistic response of 3.9 μm upon exposure to zinc. The residues highly plausible to interact with the His-substituted positions were subsequently replaced by Ala which is unable to bind zinc. Strikingly, the double mutant MC4R-I104H/D122A was unable to respond to zinc, whereas the double mutant MC4R-I104H/E100A showed an agonistic response to zinc at 0.7 μm (Fig. 2A). This indicates that I104H is forming an agonistic interhelical zinc site with Asp122, connecting TM2 and TM3 horizontally. Since MC4R-I104H/E100A could respond to zinc, the possibility of a solitary intrahelical site was unlikely. It was evident that the agonistic response derived by the single mutant MC4R-I125H was due to Glu100 (TM2) since the double mutant MC4R-E100A/I125H was unable to respond to zinc. There was also a possibility for an intrahelical interaction between I125H (TM3) and Asp122 (TM3). However, this did not appear to be the case since the double mutant MC4R-I125H/D122A responded to zinc with a potency of 2.2 μm (Fig. 2B). Taken together this shows that we have created two separate interhelical agonistic binding sites for zinc between TM2 and TM3 in the human MC4R. This clearly shows that this area of the receptor is crucial for activation, and t" @default.
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