Conversion of agonist site to metal-ion chelator site in the β ₂ -adrenergic receptor

Abstract

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the ␤2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu 2؉ , and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structureactivity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will. R hodopsin-like G protein-coupled receptors, which constitute the largest superfamily of proteins in our organism, are believed to have a common seven helical transmembrane (7TM) fold and conceivably a common, still elusive molecular mechanism of activation (1). Cryoelectron microscopy of rhodopsin has provided the basis for a generally accepted model for the overall structure of 7TM receptors (2-5). However, their detailed three-dimensional structure and the conformational changes leading to their activation is still rather ill-defined (6-8).Previously metal-ion site engineering has been employed for structural and functional analysis of 7TM receptors (9-12). Originally, the binding site for a nonpeptide antagonist was exchanged with a high affinity, tridentate Zn 2ϩ binding site located between two helices in the NK 1 tachykinin receptor (9). This site could be transferred as a zinc switch without loss of metal-ion affinity to the distantly related -opioid receptor, indicating that the overall structure within the family of rhodopsin-like 7TM receptors is well conserved (10). Interhelical bidentate zinc sites subsequently have been used to probe helix-helix interactions and, for example to prove that the seven helical bundle is arranged in an anti-clock-wise arrangement as viewed from outside the cell (11). However, as yet all metal-ion sites created both by us and others have been antagonistic (9-13). Such sites are inte...

Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

“…This indicates that the site constructed in K260H is relatively specific for Zn 2ϩ . The effect of Cu 2ϩ (as the free ion or complexed with phenanthroline) on metal ion sites constructed in TMs of family A GPCRs have varied from being just as potent to being significantly weaker than that of that of Zn 2ϩ (41,42,44). Whether Cu 2ϩ and Co 2ϩ do not bind to K260H or whether they bind but, unlike Zn 2ϩ , are unable to induce a conformational lock on the region is impossible to determine.…”

“…In both scenarios, Zn 2ϩ binding clearly has to involve residues on two strands in the ATD lip to have any implications for the signal transduction through the receptor. Analogously, both antagonistic and agonistic zinc sites created by mutations in one TM of family A and B GPCRs have been significantly "improved" by the introduction of coordinating zinc ligands in another TM (39,(42)(43)(44)46).…”

“…The typical coordination arrangement of Zn 2ϩ binding to proteins is tetrahedral or distorted tetrahedral, where the protein most often contributes with three or four ligands to the binding (27)(28)(29). However, zinc and other metal ions are able to coordinate to proteins in a bidentate manner, with solvent molecules acting as the remaining ligands (29, 50 -52), and several bidentate zinc sites have been introduced in the TMs of GPCRs (39,40,(42)(43)(44)(45)(46). Considering that the residues involved in zinc binding to K260H mGluR1 are located in the top of the ATD lobe, the region would be expected to be solvent-accessible.…”

Molecular Interactions of Cyclam and Bicyclam Non-peptide Antagonists with the CXCR4 Chemokine Receptor

“…The two major hits for AMD3100 binding (one of which was shown to be involved in cyclam binding) were located at each end of the main ligandbinding pocket, which we previously have analyzed in great detail in several other 7TM receptors, for example, by metal ion site engineering (17)(18)(19). In a molecular model of the CXCR4 receptor built over the recently published x-ray structure of rhodopsin (20), AMD3100 could be manually docked directly in between the two proposed cyclam-binding sites.…”

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

“…Such artificial intrahelical and interhelical binding sites have been used effectively to determine the orientation and exact distances between the ␣-helices of the tachykinin, opioid, and the ␤-adrenergic receptor families (4 -6). Moreover, in two previous studies (6,7), 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 (3,8).…”