Structural basis of beta-adrenergic receptor function

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the beta-adrenergic receptor (beta AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the beta AR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the beta AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur. Structural similarities among G protein-linked receptors suggest that the information gained from the genetic analysis of the beta AR should help define functionally important regions of other receptors of this class.     

A single amino acid substitution in the beta-adrenergic receptor promotes partial agonist activity from antagonists

The family of G-protein-linked receptors includes many important pharmacological targets, of which the beta-adrenergic receptor is one of the best characterized. A better understanding of those factors that determine whether a ligand functions as an antagonist or as an agonist would facilitate the development of pharmaceutical agents that act at these receptors. Site-directed mutagenesis of the hamster beta 2-adrenergic receptor has implicated the conserved Asp113 residue in the third hydrophobic domain of the receptor in the interaction with cationic amine agonists and antagonists (Strader, C. D., Sigal, I. S., Candelore, M. R., Rands, E., Hill, W. S., and Dixon, R. A. F. (1988) J. Biol. Chem, 263, 10267-10271). We now report that substitution of Asp113 with a glutamic acid residue results in a mutant beta-adrenergic receptor which recognizes several known beta-adrenergic antagonists as partial agonists. This partial agonist activity requires the presence of a carboxylate side chain on the amino acid residue at position 113 and is not observed when an asparagine residue is substituted at this position. These observations support the existence of overlapping binding sites for agonists and antagonists on the beta-adrenergic receptor and demonstrate that genetic engineering of receptors can complement structure-activity studies of ligands in defining the molecular interactions involved in receptor activation.     

Identification of two serine residues involved in agonist activation of the beta-adrenergic receptor

Pharmacophore mapping of the ligand binding domain of the beta-adrenergic receptor has revealed specific molecular interactions which are important for agonist and antagonist binding to the receptor. Previous site-directed mutagenesis experiments have demonstrated that the binding of amine agonists and antagonists to the receptor involves an interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor (Strader, C. D., Sigal, I. S., Candelore, M. R., Rands, E., Hill, W. S., and Dixon, R. A. F. (1988) J. Biol. Chem. 263, 10267-10271). We have now identified 2 serine residues, at positions 204 and 207 in the fifth hydrophobic domain of the beta-adrenergic receptor, which are critical for agonist binding and activation of the receptor. These serine residues are conserved with G-protein-coupled receptors which bind catecholamine agonists, but not with receptors whose endogenous ligands do not have the catechol moiety. Removal of the hydroxyl side chain from either Ser204 or Ser207 by substitution of the serine residue with an alanine attenuates the activity of catecholamine agonists at the receptor. The effects of these mutations on agonist activity are mimicked selectively by the removal of the catechol hydroxyl moieties from the aromatic ring of the agonist. The data suggest that the interaction of catecholamine agonists with the beta-adrenergic receptor involves two hydrogen bonds, one between the hydroxyl side chain of Ser204 and the meta-hydroxyl group of the ligand and a second between the hydroxyl side chain of Ser207 and the para-hydroxyl group of the ligand.     

Identification of three adjacent amino acids of interleukin-2 receptor beta chain which control the affinity and the specificity of the interaction with interleukin-2.

The beta chain of the interleukin-2 (IL-2) receptor (IL-2R beta) and the interleukin-3 (IL-3) binding protein AIC2A are members of the family of cytokine receptors, which also includes the receptors for growth hormone (GHR) and prolactin. A four amino acid sequence of AIC2A has recently been shown to be critical for IL-3 binding. We analyze here the function of the analogous sequence of human IL-2R beta and identify three amino acids, Ser132, His133 and Tyr134, which play a critical role in IL-2 binding. We show that some mutant IL-2 proteins with substitutions of a critical Asp residue in the N-terminal alpha-helix bind the mutant IL-2R beta receptor with a higher affinity than the wild-type receptor. This suggests that the critical Asp34 in the ligand and the sequence Ser-His-Tyr (positions 132-134) in the receptor interact directly. On the double barrel beta-stranded structural model of cytokine receptors, the residues important for ligand binding in IL-2R beta, AIC2A and GHR map to strikingly similar locations within a barrel, with the interesting difference that it is the N-terminal barrel for GHR and the C-terminal barrel for IL-2R beta and AIC2A.     

Identification of secretin,vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln(3) side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp(3)]glucagon as well as by glucagon.     

Genetic analysis of the molecular basis for beta-adrenergic receptor subtype specificity

Pharmacological analysis of ligand binding to the beta-adrenergic receptor (beta AR) has revealed the existence of two distinct receptor subtypes (beta 1 and beta 2) which are the products of different genes. The predicted amino acid sequences of the beta 1 and beta 2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human beta 1 and hamster beta 2 receptors. Analysis of the ligand-binding characteristics of these hybrid receptors revealed that residues in the middle portion of the beta AR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacements of regions of the hamster beta 2 AR with the analogous regions from the avian beta 1 AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand-binding properties of the receptor, the subtype-specific differences in amine-substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the beta 1 and beta 2 receptors.     

A point mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor increases its affinity for a family of beta receptor antagonists.

Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.     

Substitution of ASP193 to ASN at the active site of ribulose-1,5-bisphosphate carboxylase results in conformational changes.

The crystal structure of a mutant of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillium rubrum, where Asp193, one of the ligands of the magnesium ion at the activator site, is replaced by Asn, has been determined to a nominal resolution of 0.26 nm. The mutation of Asp to Asn induces both local and global conformation changes as follows. The side chain of Asn193 moves away from the active site and interacts with main-chain oxygen of residue 165, located in the neighbouring strand beta 1 of the alpha/beta barrel. The side chain of Lys166, which forms a salt bridge with Asp193 in the wild-type enzyme, interacts with Asn54 from the second subunit and creates a new subunit-subunit interaction. Another new subunit-subunit interaction is formed, more than 1.2 nm away from the site of the mutation. In the mutant enzyme, the side chain of Asp263 interacts with the side chain of Thr106 from the second subunit. Asp193 is not part of a subunit-subunit interface area or an allosteric regulatory site. Nevertheless, replacement of this residue by Asn results, unexpectedly, in a difference in the packing of the two subunits, which can be described as a slight rotation of one of the subunits relative to the second. The observed structural changes at the active site of the enzyme provide a molecular explanation for the differing behaviour of the Asp193----Asn mutant with respect to activation.     

Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.     

3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity.

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased kcat. The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.     

Conserved aspartic acid residues 79 and 113 of the beta-adrenergic receptor have different roles in receptor function

Deletion mutagenesis experiments have demonstrated that the binding site of the beta-adrenergic receptor involves the hydrophobic core of the protein (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). Single amino acid replacements for the conserved Asp79 and Asp113 within this putative transmembrane region had profound effects on the ability of the receptor to bind radiolabeled ligands (Strader, C. D., Sigal, I. S., Register, R. B., Candelore, M. R., Rands, E., and Dixon, R. A. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4384-4388). In this report we have analyzed the ability of these mutant receptors to stimulate adenylyl cyclase in the presence of agonists. The substitution of Asp79 with Ala caused 10-fold increases in both the Kd for isoproterenol binding and the Kact for adenylyl cyclase stimulation. The substitution of Asp113 by Asn or Glu resulted in 8,000-40,000 and 300-1,500-fold increases, respectively, in the Kact values for agonist stimulation of adenylyl cyclase without altering the maximum level of stimulation. Whereas the binding of antagonists to the receptor was not affected by substitution of Asp79, substitution of Asp113 decreased the affinity for the antagonist propranolol by 10,000-fold. These data are consistent with overlapping but not identical binding sites for agonists and antagonists on the beta-adrenergic receptor, in which the carboxylate group of Asp113 interacts with the amino group of the ligand. The sequence similarity among the family of G-protein-linked receptors suggests that the presence of an Asp residue at the analogous position of one of these receptors is predictive of the ability of the receptor to bind amines as ligands.     

Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system

While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).     

Molecular dynamics simulations of wild type and mutant of Pin1 peptidyl-prolyl isomerase

Pin1 catalyses the intrinsically slow process of cis-trans isomerisation and has been identified as a possible drug target in many diseases. Recently, the wild type (WT) and the Cys113Asp mutant of the Pin1 peptidyl-prolyl isomerase (PPIase) domain were determined by nuclear magnetic resonance. In this article, the WT and Cys113Asp mutant of PPIase domain are studied by molecular dynamics simulations. The structural stability analysis shows that the Cys113Asp mutation leads to the higher fluctuation of hydrophobic core in PPIase domain. The intrinsic correlated motions are important for the catalytic function of Pin1, whereas the Cys113Asp mutant system loses pivotal dynamical properties and develops wider conformational states than those in WT system. The intramolecular hydrogen bonds play crucial roles in the structural stability of PPIase domain. The mutated residue Asp113 attracts the side chain of His59 in the Cys113Asp system, which unbalances the internal interactions inside the catalytic tetrad. Meanwhile, the conformational changes of PPIase domain affect the side chain orientations of Lys63 and Arg69, which limit their binding with substrates. The Cys113Asp mutation destabilises the whole binding region of Pin1 PPIase domain, so the catalysis activity is severely reduced. These results are consistent with experimental studies and may help to understand the isomerisation mechanisms of Pin1.     

Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction

Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2. 61(98))Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His(2) residue. This mutant showed smaller decreases in affinity for His(2)-substituted ligands. The loss of preference for His(2)-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His(2). Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the delta NH group of His(2) and that Asp(2. 61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His(2) of GnRH that helps stabilize agonist complexing.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Chemical-scale studies on the role of a conserved aspartate in preorganizing the agonist binding site of the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor and related Cys-loop receptors are ligand-gated ion channels that mediate fast synaptic transmission throughout the central and peripheral nervous system. A highly conserved aspartate residue (D89) that is near the agonist binding site but does not directly contact the ligand plays a critical part in receptor function. Here we probe the role of D89 using unnatural amino acid mutagenesis coupled with electrophysiology. Homology modeling implicates several hydrogen bonds involving D89. We find that no single hydrogen bond is essential to proper receptor function. Apparently, the side chain of D89 establishes a redundant network of hydrogen bonds; these bonds preorganize the agonist binding site by positioning a critical tryptophan residue that directly contacts the ligand. Earlier studies of the D89N mutant led to the proposal that a negative charge at this position is essential for receptor function. However, we find that receptors with neutral side chains at position 89 can function well, if the side chain is less perturbing than the amide of asparagine (nitro or keto groups allow function) or if a compensating backbone mutation is introduced to relieve unfavorable electrostatics.     

Zinc binding and dimerization of Streptococcus pyogenes pyrogenic exotoxin C are not essential for T-cell stimulation

Streptococcal pyrogenic enterotoxin C (Spe-C) is a superantigen virulence factor produced by Streptococcus pyogenes that activates T-cells polyclonally. The biologically active form of Spe-C is thought to be a homodimer containing an essential zinc coordination site on each subunit, consisting of the residues His(167), His(201), and Asp(203). Crystallographic data suggested that receptor specificity is dependent on contacts between the zinc coordination site of Spe-C and the beta-chain of the major histocompatibility complex type II (MHCII) molecule. Our results indicate that only a minor fraction of dimer is present at T-cell stimulatory concentrations of Spe-C following mutation of the unpaired side chain of cysteine at residue 27 to serine. Mutations of amino acid residues His(167), His(201), or Asp(203) had only minor effects on protein stability but resulted in greatly diminished MHCII binding, as measured by surface plasmon resonance with isolated receptor/ligand pairs and flow cytometry with MHCII-expressing cells. However, with the exception of the mutants D203A and D203N, mutation of the zinc-binding site of Spe-C did not significantly impact T-cell activation. The mutation Y76A, located in a polar pocket conserved among most superantigens, resulted in significant loss of T-cell stimulation, although no effect was observed on the overall binding to human MHCII molecules, perhaps because of the masking of this lower affinity interaction by the dominant zinc-dependent binding. To a lesser extent, mutations of side chains found in a second conserved MHCII alpha-chain-binding site consisting of a hydrophobic surface loop decreased T-cell stimulation. Our results demonstrate that dimerization and zinc coordination are not essential for biological activity of Spe-C and suggest the contribution of an alternative MHCII binding mode to T-cell activation.     

Roles of the human Rad51 L1 and L2 loops in DNA binding

The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.