金黄色葡萄球菌agr系统受体蛋白AgrC的跨膜结构分析析

金黄色葡萄球菌agr 系统中,受体组氨酸蛋白激酶AgrC 能够被同源或非同源自诱导信号分子(autoinducing peptides,AIPs) 激活或抑制。通过TMHMM软件对四种agr 型AgrC蛋白的氨基酸序列进行分析及跨膜结构预测,发现agr玉型和agr郁型AgrC蛋白可能在胞外的第二个Loop 环与其AIP相互作用,agr域型可能与AIP域在胞外的第一个或第二个Loop 环相互作用。     

Ligand-receptor recognition for activation of quorum sensing in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The accessory gene regulator (agr) locus controls many of the virulence toxins involved in Staphylococcus aureus pathogenesis, and can be divided into four specificity groups. AgrC is the only group-specific receptor to mediate both intra-group activation and inter-group inhibition. We studied the ligand-receptor recognition of the agr system in depth by using a luciferase reporter system to identify the key residues responsible for AgrC activation in two closely related agr groups, AgrC-I, and AgrC-IV. Fusion PCR and site-directed mutagenesis were used to screen for functional residues of AgrC. Our data suggest that for AgrC-IV activation, residue 101 is critical for activating the receptor. In contrast, the key residues for the activation of AgrC-I are located at residues 49∼59, 107, and 116. However, three residue changes, T101A, V107S, I116S, are sufficient to convert the AIP recognizing specificity from AgrC-IV to AgrC-I.     

Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC

Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator ( agr ) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans- autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection.     

Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated upon binding of a self-coded autoinducing peptide (AIP) to the receptor-histidine kinase, AgrC. The AIPs consist of a thiolactone macrocyle and an exocyclic "tail", both of which are important for function. In this report, characterization of the unique AIPs from the four known agr specificity groups of Staphylococcus aureus has been completed, along with analysis of cross-group inhibition of AgrC activation by each of the four AIPs. The following conclusions have been drawn: (i) The native thiolactone macrocyle and tail are necessary and sufficient for full activation by the AIPs, whereas the AIP-I macrocycle alone is a partial agonist. (ii) The native N-terminus is less critical, as that of AIP-I can be modified without affecting bioactivity, although that of AIP-III cannot. (iii) The ring and tail may function differently in different AIPs. Thus the group I and IV AIPs differ at a single (endocyclic) residue, which is the determinant of AIP specificity for these two groups and is essential for function. A similarly critical residue in AIP-II, however, is exocyclic. (iv) Cross-inhibition is more tolerant of sequence and structural diversity than is activation, suggesting that the AIPs interact differently with cognate than with heterologous receptors. (v) Chimeric peptides, in which the tails and macrocycles are switched, do not activate and instead inhibit receptor activation. These data suggest a model in which activation and inhibition involves different binding orientations within the ligand binding pocket of each receptor.     

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the α-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.     

Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.     

Structure, activity and evolution of the group I thiolactone peptide quorum-sensing system of Staphylococcus aureus

In Staphylococcus aureus, the agr locus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including the L-alanine and D-amino acid scanned peptides) was synthesized to determine the functionally critical residues within the S. aureus group I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activate agr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite for agr activation. Although each of the D-amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C-terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinical S. aureus isolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group I S. aureus strains, the producer strains possess a functional agr locus dependent on the endogenous peptide and, as such, constitute a fourth S. aureus AIP pheromone group (group IV). The addition of exogenous synthetic AIPs to S. aureus inhibited the production of toxic shock syndrome toxin (TSST-1) and enterotoxin C3, confirming the potential of quorum-sensing blockade as a therapeutic strategy.     

Reversible and specific extracellular antagonism of receptor-histidine kinase signaling.

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling.     

Site-directed mutagenesis of beta-adrenergic receptors. Identification of conserved cysteine residues that independently affect ligand binding and receptor activation

Using site-directed mutagenesis of the human beta 2-adrenergic receptor and continuous expression in B-82 cells, the role of 3 conserved cysteines in transmembrane domains and 2 conserved cysteines in the third extracellular domain in receptor function was examined. Cysteine was replaced with serine in each mutant receptor as this amino acid is similar to cysteine in size but it cannot form disulfide linkages. Replacement of cysteine residues 77 and 327, in the second and seventh transmembrane-spanning domains, respectively, had no effect on ligand binding or the ability of the receptor to mediate isoproterenol stimulation of adenylate cyclase. Substitution of cysteine 285, in the sixth transmembrane domain of the receptor, produced a mutant receptor with normal ligand-binding properties but a significantly attenuated ability to mediate stimulation of adenylate cyclase. Mutation of cysteine residues 190 and 191, in the third extracellular loop of the beta 2 receptor, had qualitatively similar effects on ligand binding and isoproterenol-mediated stimulation of adenylate cyclase. Replacement of either of these residues with serine produced mutant receptors that displayed a marked loss in affinity for both beta-adrenergic agonists and antagonists. Replacement of both cysteine 190 and 191 with serine had an even greater effect on the ability of the receptor to bind ligands. Consistent with the loss of Ser190 and/or Ser191 mutant receptor affinity for agonists was a corresponding shift to the right in the dose-response curve for isoproterenol-induced increases in intracellular cyclic AMP concentrations in cells expressing the mutant receptors. These data implicate one of the conserved transmembrane cysteine residues in the human beta 2-adrenergic receptor in receptor activation by agonists and also suggest that conserved cysteine residues in an extracellular domain of the receptor may be involved in ligand binding.     

Biosynthesis of Staphylococcus aureus autoinducing peptides by using the synechocystis DnaB mini-intein

The Agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from Synechocystis sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against S. aureus. The AIP activity was confirmed through Agr interference and activation profiling with developed S. aureus reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of S. aureus AIPs for many biological applications.     

Structural basis for ligand recognition and discrimination of a quorum-quenching antibody

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using “quorum sensing,” which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu^H95 provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.     

Role of the first extracellular loop in the functional activation of CCR2. The first extracellular loop contains distinct domains necessary for both agonist binding and transmembrane signaling.

The physiological cellular responses to monocyte chemoattractant protein-1 (MCP-1), a potent chemotactic and activating factor for mononuclear leukocytes, are mediated by specific binding to CCR2. The aim of this investigation is to identify receptor microdomains that are involved in high affinity agonist binding and receptor activation. The results from our functional studies in which we utilized neutralizing antisera against CCR2 are consistent with a multidomain binding model, previously proposed by others. The first extracellular loop was of particular interest, because in addition to a ligand-binding domain it contained also information for receptor activation, crucial for transmembrane signaling. Replacement of the first extracellular loop of CCR2 with the corresponding region of CCR1 decreased the MCP-1 binding affinity about 10-fold and prevented transmembrane signaling. A more detailed analysis by site-directed mutagenesis revealed that this receptor segment contains two distinct microdomains. The amino acid residues Asn(104) and Glu(105) are essential for high affinity agonist binding but are not involved in receptor activation. In contrast, the charged amino acid residue His(100) does not contribute to ligand binding but is vital for receptor activation and initiation of transmembrane signaling. We hypothesize that the interaction of agonist with this residue initiates the conformational switch that allows the formation of the functional CCR2-G protein complex.     

New insight into transmembrane topology of Staphylococcus aureus histidine kinase AgrC

Staphylococcus aureus accessory gene regulator (agr) locus controls the expression of virulence factors through a classical two-component signal transduction system that consists of a receptor histidine protein kinase AgrC and a cytoplasmic response regulator AgrA. An autoinducing peptide (AIP) encoded by agr locus activates AgrC, which transduces extracellular signals into the cytoplasm. Despite extensive investigations to identify AgrC–AIP interaction sites, precise signal recognition mechanisms remain unknown. This study aims to clarify the membrane topology of AgrC by applying the green fluorescent protein (GFP) fusion technique and the substituted cysteine accessibility method (SCAM). However, our findings were inconsistent with profile obtained previously by alkaline phosphatase. We report the topology of AgrC shows seven transmembrane segments, a periplasmic N-terminus, and a cytoplasmic C-terminus.     

Identification of a domain of ETA receptor required for ligand binding.

Various chimeric ETA and ETB receptors were produced in CHO cells for the elucidation of a specific domain which influences the affinity of the receptor toward BQ-123, a selective ETA antagonist. Replacement of the first extracellular loop domain (B-loop) of the ETA receptor with the corresponding domain of the ETB receptor, reduced the inhibition by BQ-123 drastically, while the replacements of other extracellular domains of ETA did not. By contrast, the introduction of the B-loop of ETA in place of the corresponding domain of the ETB receptor endowed the ETB-based chimeric receptor with a sensitivity to BQ-123. These observations suggest that the B-loop domain of the ETA receptor is involved in ligand binding.     

Interspecies molecular chimeras of kit help define the binding site of the stem cell factor.

The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.     

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.     

Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus

The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC , to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr⁺ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr⁺ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus , and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.     

Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.     

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.