An Indel in Transmembrane Helix 2 Helps to Trace the Molecular Evolution of Class A G-Protein-Coupled Receptors

Class A G-protein-coupled receptors (GPCRs) constitute a large family of transmembrane receptors. Helical distortions play a major role in the overall fold of these receptors. Most are related to conserved proline residues. However, in transmembrane helix 2, the proline pattern is not conserved, and when present, proline may be located at position 2.58, 2.59, or 2.60. Sequence analysis, three-dimensional data mining, and molecular modeling were undertaken to investigate the origin of this unusual pattern. Taken together, the data strongly support the assumption that an indel led to two structural motifs for helix 2: a bulged structure in P2.59 and P2.60 receptors and a “typical” proline kink in P2.58 receptors. The proline pattern of helix 2 can be used as an evolutionary marker and helps to trace the molecular evolution of class A GPCRs. Two indel events yielding functional receptors occurred independently. One indel arose very early in GPCR evolution, in a bilaterian ancestor, before the protostome-deuterostome divergence. This indel led to the split between the P2.58 somatostatin/opioid receptors and other peptide receptors with the P2.59 pattern. A second indel also occurred in insect opsins and corresponds to a deletion. Subfamilies with proline at position 2.59 or no proline expanded earlier, whereas P2.60 receptors remained marginal throughout evolution. P2.58 receptors underwent rapid expansion in vertebrates with the development of the chemokine and purinergic receptor subfamilies from somatostatin/opioid-related ancestors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Syncytium-inducing mutations localize to two discrete regions within the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B.

Herpes simplex virus type 1 glycoprotein B (gB) is essential for virus entry, an event involving fusion of the virus envelope with the cell surface membrane, and virus-induced cell-cell fusion, resulting in polykaryocyte, or syncytium, formation. The experiments described in this report employed a random mutagenesis strategy to develop a more complete genetic map of mutations resulting in the syn mutant phenotype. The results indicate that syn mutations occur within two essential and highly conserved hydrophilic, alpha-helical regions of the gB cytoplasmic domain. Region I is immediately proximal to the transmembrane domain and includes residues R796 to E816/817. Region II is localized centrally in the cytoplasmic domain and includes residues A855 and R858. Positively charged residues were particularly affected in both regions, suggesting that charge interactions may be required to suppress the syn mutant phenotype. No syn mutations were identified within the transmembrane domain. A virus containing a rate of entry (roe) mutation at residue A851, either within or immediately proximal to syn region II, was isolated. Since roe mutations have also been discovered in the external domain of gB, it appears likely that the external and cytoplasmic domains cooperate in virus penetration. Moreover, the observation that both roe and syn mutations occur in the cytoplasmic domain further suggests that gB functions in an analogous manner in both membrane fusion events. It might be predicted from these observations that membrane fusion involves transduction of a fusion signal along the gB molecule through the transmembrane domain. Communication between the external and cytoplasmic domain may thus be required for gB-mediated membrane fusion events.     

NMR solution structure and receptor peptide binding of the CC chemokine eotaxin-2

The human CC chemokine eotaxin-2 is a specific agonist for the chemokine receptor CCR3 and may play a role in the recruitment of eosinophils in allergic diseases and parasitic infections. We report the solution structure of eotaxin-2 determined using heteronuclear and triple resonance NMR methods. A family of 20 structures was calculated by hybrid distance geometry-simulated annealing from 854 NOE distance restraints, 48 dihedral angle restraints, and 12 hydrogen bond restraints. The structure of eotaxin-2 (73 amino acid residues) consists of a helical turn (residues 17-20) followed by a 3-stranded antiparallel beta-sheet (residues 22-26, 37-41, and 44-49) and an alpha-helix (residues 54-66). The N-loop (residues 9-16) is packed against both the sheet and the helix with the two conserved disulfide bonds tethering the N-terminal/N-loop region to the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 7-66) are 0.52 and 1.13 A, respectively. A linear peptide corresponding to the N-terminal region of CCR3 binds to eotaxin-2, inducing concentration-dependent chemical shift changes or line broadening of many residues. The distribution of these residues suggests that the peptide binds into an extended groove located at the interface between the N-loop and the beta2-beta3 hairpin. The receptor peptide may also interact with the N-terminus of the chemokine and part of the alpha-helix. Comparison of the eotaxin-2 structure with those of related chemokines indicates several structural features that may contribute to receptor specificity.     

Generation of an isogenic collection of yeast actin mutants and identification of three interrelated phenotypes

A large collection of yeast actin mutations has been previously isolated and used in numerous studies of actin cytoskeletal function. However, the various mutations have been in congenic, rather than isogenic, backgrounds, making it difficult to compare the subtle phenotypes that are characteristic of these mutants. We have therefore placed 27 mutations in an isogenic background. We used a subset of these mutants to compare the degree to which different actin alleles are defective in sporulation, endocytosis, and growth on NaCl-containing media. We found that the three phenotypes are highly correlated. The correlations are specific and not merely a reflection of general growth defects, because the phenotypes are not correlated with growth rates under normal conditions. Significantly, those actin mutants exhibiting the most severe phenotypes in all three processes have altered residues that cluster to a small region of the actin crystal structure previously defined as the fimbrin (Sac6p)-binding site. We examined the relationship between endocytosis and growth on salt and found that shifting wild-type or actin mutant cells to high salt reduces the rate of alpha-factor internalization. These results suggest that actin mutants may be unable to grow on salt because of additive endocytic defects (due to mutation and salt).     

Identification of CC chemokine receptor 7 residues important for receptor activation

The binding pocket of family A GPCRs that bind small biogenic amines is well characterized. In this study we identify residues on CC chemokine receptor 7 (CCR-7) that are involved in agonist-mediated receptor activation but not in high affinity ligand binding. The mutations also affect the ability of the ligands to induce chemotaxis. Two of the residues, Lys3.33(137) and Gln5.42(227), are consistent with the binding pocket described for biogenic amines, while Lys3.26(130) and Asn7.32(305), are found at, or close to, the cell surface. Our observations are in agreement with findings from other peptide and chemokine receptors, which indicate that receptors that bind larger ligands contain contact sites closer to the cell surface in addition to the conventional transmembrane binding pocket. These findings also support the theory that chemokine receptors require different sets of interactions for high affinity ligand binding and receptor activation.     

Determinants of the factor IX mutational spectrum in haemophilia B: an analysis of missense mutations using a multi-domain molecular model of the activated protein

A multi-domain molecular model of factor IXa was constructed by comparative methods. The quaternary structure of the protein was assembled by docking individual domains through consideration of their shape complementarity, polaric properties and the location of cross-reacting material positive/negative (CRM^+/–) variants on domain surfaces. Some 217 different missense mutations in the factor IX (F9) gene were then selected for study. Using maximum likelihood analysis, missense mutations affecting highly conserved amino acid residues of factor IX were shown to be 15–20 times more likely to result in haemophilia B than those affecting non-conserved residues. However, about one quarter of this increase in likelihood of clinical observation could be attributed to the magnitude of the amino acid exchange. Missense mutations in structurally conserved residues were found to be 2.1-fold more likely to come to clinical attention than those in structurally variable residues. Missense mutations in residues whose side chains were inwardly pointing were 3.6-fold more likely to be observed than those in surface residues. These observations imply a complex hierarchy of sequence/structure conservation in the protein. The severity of the clinical phenotype correlated with both the extent of the evolutionary sequence conservation of the residue at the site of mutation and the magnitude of the amino acid exchange. Further, the substitution of residues exhibiting minimal side chain solvent accessibility was associated disproportionately with severe haemophilia compared with that of surface residues. Clusters of CRM⁺ mutations were observed at factor IX-specific residues on the surface of the molecule. These clusters may reflect factor IX-specific docking interactions. The likelihood that a given factor IX mutation will come to clinical attention is therefore a complex function of the sequence characteristics of the F9 gene, the nature of the amino acid substitution, its precise location and immediate environment within the protein molecule, and its resulting effects on the structure and function of the protein.This paper is dedicated to the memory of Andrew Wacey     

Identification of surface residues of the monocyte chemotactic protein 1 that affect signaling through the receptor CCR2

The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.     

Proton NMR and CD solution conformation determination and opioid receptor binding studies of a dynorphin A(1-17) model peptide

The opioid peptide dynorphin A(1-17) contains a peptide segment in residues 7-15 with the potential to form an amphiphilic beta-strand. This amphiphilic structure may, like the amphiphilic alpha-helices found in many other peptide hormones, be an important determinant of its interactions with membranes and receptors. In order to investigate and characterize these interactions, we have synthesized a 17-residue dynorphin analogue (YGGFLKKVKPKVKVKSS) that incorporates a peptide model of this amphiphilic secondary structure with minimized homology (25%) relative to the native sequence. This peptide exhibits the full biological potency of dynorphin in assays of kappa-opioid receptor binding, and is more selective for this type of opioid receptor than the natural peptide. The conformation of the model peptide in aqueous solution has been investigated in detail by NMR spectroscopy. The values of the NH-CH alpha coupling constants together with rotating frame NOEs indicate the presence of an amphiphilic structure together with some beta-strand structure in residues 7-15, and demonstrate that a peptide model that stabilizes this structure in aqueous solution and enhances kappa-opioid receptor selectivity can be successfully designed using using alternating lysine and valine residues.     

Chemotype-selective Modes of Action of κ-Opioid Receptor Agonists

The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1–17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx. Minimally biased docking of the tested ligands into the antagonist-bound KOR structure generated distinct binding modes, which were then evaluated biochemically and pharmacologically. Our analysis identified two types of mutations: those that affect receptor function primarily via ligand binding and those that primarily affect function. The shared and differential mechanisms of agonist binding and activation in KOR are further discussed. Usually, mutations affecting function more than binding were located at the periphery of the binding site and did not interact strongly with the various ligands. Analysis of the crystal structure along with the present results provide fundamental insights into the activation mechanism of the KOR and suggest that “functional” residues, along with water molecules detected in the crystal structure, may be directly involved in transduction of the agonist binding event into structural changes at the conserved rotamer switches, thus leading to receptor activation.     

Identification and characterization of opioid and somatostatin binding sites in the opossum kidney (OK) cell line and their effect on growth

Opioids and somatostatin analogs have been implicated in the modulation of renal water handling, but whether their action is accomplished through central and/or peripheral mechanisms remains controversial. In different cell systems, on the other hand, opioids and somatostatin inhibit cell proliferation. In the present study, we have used an established cell line, derived from opossum kidney (OK) proximal tubules, in order to characterize opioid and somatostatin receptors and to investigate the action of opioids and somatostatin on tubular epithelial tissue. Our results show the presence of one class of opioid binding sites with kappa₁ selectivity (K_D 4.6 ± 0.9 nM, 57,250 sites/cell), whereas delta, mu, or other subtypes of the kappa site were absent. Somatostatin presents also a high affinity site on these cells (K_D 24.5 nM, 330,000 sites/cell). No effect of either opioids or somatostatin on the activity of the Na⁺/P_i cotransporter was observed, indicating that these agents do not affect ion transport mechanisms. However, opioid agonists and somatostatin analogs decrease OK cell proliferation in a dose-dependent manner; in the same nanomolar concentration range, they displayed reversible specific binding for these agents. The addition of diprenorphine, a general opioid antagonist, reversed the effects of opioids, with the exception of morphine. Furthermore, morphine interacts with the somatostatin receptor in this cell line too, as was the case in the breast cancer T47D cell line. Our results indicate that in the proximal tubule opioids and somatostatin do not affect ion transport, but they might have a role in the modulation of renal cell proliferation either during ontogenesis or in kidney repair. © 1996 Wiley-Liss, Inc.     

Filling small, empty protein cavities: structural and energetic consequences

Most proteins contain small cavities that can be filled by replacing cavity-lining residues by larger ones. Since shortening mutations in hydrophobic cores tend to destabilize proteins, it is expected that cavity-filling mutations may conversely increase protein stability. We have filled three small cavities in apoflavodoxin and determined by NMR and equilibrium unfolding analysis their impact in protein structure and stability. The smallest cavity (14 A3) has been filled, at two different positions, with a variety of residues and, in all cases, the mutant proteins are locally unfolded, their structure and energetics resembling those of an equilibrium intermediate of the thermal unfolding of the wild-type protein. In contrast, two slightly larger cavities of 20 A3 and 21 A3 have been filled with Val to Ile or Val to Leu mutations and the mutants preserve both the native fold and the equilibrium unfolding mechanism. From the known relationship, observed in shortening mutations, between stability changes and the differential hydrophobicity of the exchanged residues and the volume of the cavities, the filling of these apoflavodoxin cavities is expected to stabilize the protein by approximately 1.5 kcal mol(-1). However, both urea and thermal denaturation analysis reveal much more modest stabilizations, ranging from 0.0 kcal mol(-1) to 0.6 kcal mol(-1), which reflects that the accommodation of single extra methyl groups in small cavities requires some rearrangement, necessarily destabilizing, that lowers the expected theoretical stabilization. As the size of these cavities is representative of that of the typical small, empty cavities found in most proteins, it seems unlikely that filling this type of cavities will give rise to large stabilizations.     

Analysis of correlated mutations in HIV-1 protease using spectral clustering

MOTIVATION: The ability of human immunodeficiency virus-1 (HIV-1) protease to develop mutations that confer multi-drug resistance (MDR) has been a major obstacle in designing rational therapies against HIV. Resistance is usually imparted by a cooperative mechanism that can be elucidated by a covariance analysis of sequence data. Identification of such correlated substitutions of amino acids may be obscured by evolutionary noise. RESULTS: HIV-1 protease sequences from patients subjected to different specific treatments (set 1), and from untreated patients (set 2) were subjected to sequence covariance analysis by evaluating the mutual information (MI) between all residue pairs. Spectral clustering of the resulting covariance matrices disclosed two distinctive clusters of correlated residues: the first, observed in set 1 but absent in set 2, contained residues involved in MDR acquisition; and the second, included those residues differentiated in the various HIV-1 protease subtypes, shortly referred to as the phylogenetic cluster. The MDR cluster occupies sites close to the central symmetry axis of the enzyme, which overlap with the global hinge region identified from coarse-grained normal-mode analysis of the enzyme structure. The phylogenetic cluster, on the other hand, occupies solvent-exposed and highly mobile regions. This study demonstrates (i) the possibility of distinguishing between the correlated substitutions resulting from neutral mutations and those induced by MDR upon appropriate clustering analysis of sequence covariance data and (ii) a connection between global dynamics and functional substitution of amino acids.     

Three-dimensional structure of the argininosuccinate lyase frequently complementing allele Q286R.

Argininosuccinate lyase (ASL) catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, a reaction involved in the biosynthesis of arginine in all species and in the production of urea in ureotelic species. In humans, mutations in the enzyme result in the autosomal recessive disorder argininosuccinic aciduria. Intragenic complementation has been demonstrated to occur at the ASL locus, with two distinct classes of ASL-deficient strains having been identified, the frequent and high-activity complementers. The frequent complementers participate in the majority of the complementation events observed and were found to be either homozygous or heterozygous for a glutamine to arginine mutation at residue 286. The three-dimensional structure of the frequently complementing allele Q286R has been determined at 2.65 A resolution. This is the first high-resolution structure of human ASL. Comparison of this structure with the structures of wild-type and mutant duck delta1 and delta2 crystallins suggests that the Q286R mutation may sterically and/or electrostatically hinder a conformational change in the 280's loop (residues 270-290) and domain 3 that is thought to be necessary for catalysis to occur. The comparison also suggests that residues other than R33, F333, and D337 play a role in maintaining the structural integrity of domain 1 and reinforces the suggestion that residues 74-89 require a particular conformation for catalysis. The electron density has enabled the structure of residues 6-18 to be modeled for the first time. Residues 7-9 and 15-18 are in type IV beta-turns and are connected by a loop. The conformation observed is stabilized, in part, by a salt bridge between the side chains of R12 and D18. Although the disease causing mutation R12Q would disrupt this salt bridge, it is unclear why this mutation has such a significant effect on the catalytic activity as residues 1-18 are disordered in all other delta-crystallin structures determined to date.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

A role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor

Several studies have proposed that angiotensin II (Ang II) binds to its receptor AT1 through interactions with residues in helices V and VI, suggesting that the distance between these helices is crucial for ligand binding. Based on a 3D model of AT1 in which the C-terminus of Ang II is docked, we identified the hydrophobic residues of TM V and VI pointing towards the external face of the helices, which may play a role in the structure of the binding pocket and in the structural integrity of the receptor. We performed a systematic mutagenesis study of these residues and examined the binding, localization, maturation, and dimerization of the mutated receptors. We found that mutations of hydrophobic residues to alanine in helix V do not alter binding, whereas mutations to glutamate lead to loss of binding without a loss in cell surface expression, suggesting that the external face of helix V may not directly participate in binding, but may rather contribute to the structure of the binding pocket. In contrast, mutations of hydrophobic residues to glutamate in helix VI lead to a loss in cell surface expression, suggesting that the external surface of helix VI plays a structural role and ensures correct folding of the receptor.     

Mutations in calpain 3 associated with limb girdle muscular dystrophy: analysis by molecular modeling and by mutation in m-calpain

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.     

Solution structure and dynamics of myeloid progenitor inhibitory factor-1 (MPIF-1), a novel monomeric CC chemokine

MPIF-1, a CC chemokine, is a specific inhibitor of myeloid progenitor cells and is the most potent activator of monocytes. The solution structure of myeloid progenitor inhibitor factor-1 (MPIF-1) has been determined by NMR spectroscopy. The structure reveals that MPIF-1 is a monomer with a well defined core except for termini residues and adopts the chemokine fold of three beta-strands and an overlying alpha-helix. In addition to the four cysteines that characterize most chemokines, MPIF-1 has two additional cysteines that form a disulfide bond. The backbone dynamics indicate that the disulfide bonds and the adjacent residues that include the functionally important N-terminal and N-terminal loop residues show significant dynamics. MPIF-1 is a highly basic protein (pI >9), and the structure reveals distinct positively charged pockets that could be correlated to proteoglycan binding. MPIF-1 is processed from a longer proprotein at the N terminus and the latter is also functional though with reduced potency, and both proteins exist as monomers under a variety of solution conditions. MPIF-1 is therefore unique because longer proproteins of all other chemokines oligomerize in solution. The MPIF-1 structure should serve as a template for future functional studies that could lead to therapeutics for preventing chemotherapy-associated myelotoxicity.     

Interactions between opioid and chemokine receptors: heterologous desensitization

The opioid and chemokine receptors are both members of the seven transmembrane G protein-coupled receptor (GPCR) superfamily. Desensitization is believed to be a major element of the regulation of the function of these receptors, and recent findings suggest that both agonist-dependent (homologous) desensitization and heterologous desensitization can control receptor activity. The cross-desensitization between opioid and chemokine receptors has significant implications for our understanding of both the regulation of leukocyte trafficking, as well as the regulation of chemokine receptor function in inflammatory disease states. We also review findings which suggest that pro-inflammatory chemokine receptor-induced heterologous desensitization of opioid receptors has important implications for the regulation of opioid receptor function in the nervous system.     

N-terminal domain of eotaxin-3 is important for activation of CC chemokine receptor 3

Eotaxin-3 belongs to the CC chemokine family, and specifically recognizes CC chemokine receptor (CCR) 3 that is expressed on eosinophils, basophils and helper T type 2 cells. The three-dimensional structure of eotaxin-3 determined by nuclear magnetic resonance has revealed that the N-terminal nine residues preceding the first cysteine comprise an unstructured domain, which is also observed in other chemokine molecules. In order to determine the function of the N-terminal domain of eotaxin-3, we constructed various N-terminal-deletion mutants, and then examined their binding and chemotactic activities toward eosinophils in vitro. Competitive binding studies showed that the binding affinity of truncated mutant toward CCR3 was almost the same as that of wild-type eotaxin-3 even though the N-terminal truncation involved the first through to the ninth residues. In contrast, the chemotactic activity gradually decreased with extension of the N-terminal deletion, and when the deletion extended to the eighth residue, the activity was not detected at all. Thus, the N-terminal nine residues are not critical for binding but the N-terminal eight residues are essential for activation of CCR3. The truncated eotaxin-3 proteins lacking the N-terminal eight or nine residues inhibited the chemotactic activity of chemokines that recognize CCR3. The truncated mutants can possibly be used for anti-allergic and anti-HIV-1 therapy.     

Switching agonist/antagonist properties of opiate alkaloids at the delta opioid receptor using mutations based on the structure of the orphanin FQ receptor

In an earlier study, we have demonstrated that by mutating five amino acid residues to those conserved in the opioid receptors, the OFQ receptor could be converted to a functional receptor that bound many opioid alkaloids with nanomolar affinities. Surprisingly, when the reciprocal mutations, Lys-214 --> Ala (TM5), Ile-277 --> Val/His-278 --> Gln/Ile-279 --> Val (TM6), and Ile-304 --> Thr (TM7), are introduced in the delta receptor, neither the individual mutations nor their various combinations significantly reduce the binding affinities of opioid alkaloids tested. However, these mutations cause profound alterations in the functional characteristics of the mutant receptors as measured in guanosine 5'-3-O-(thio)triphosphate binding assays. Some agonists become antagonists at some constructs as they lose their ability to activate them. Some alkaloid antagonists are transformed into agonists at other constructs, but their agonistic effects can still be blocked by the peptide antagonist TIPP. Even the delta inverse agonist 7-benzylidenenaltrexone becomes an agonist at the mutant containing both the Ile-277 --> Val/His-278 --> Gln/Ile-279 --> Val and Ile-304 --> Thr mutations. Thus, although the mutated residues are thought to be part of the binding pocket, they are critically involved in the control of the delta receptor activation process. These findings shed light on some of the structural bases of ligand efficacy. They are also compatible with the hypothesis that a ligand may achieve high affinity binding in several different ways, each having different effects on receptor activation.