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1.
Nat Med ; 13(6): 725-9, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17515896

RESUMO

Exotoxins of Staphylococcus aureus belong to a family of bacterial proteins that act as superantigens by activating a large subset of the T-cell population, causing massive release of inflammatory cytokines. This cascade can ultimately result in toxic shock syndrome and death. Therapeutics targeting the early stage of the pathogenic process, when the superantigen binds to its receptor, could limit the severity of disease. We engineered picomolar binding affinity agents to neutralize the potent toxin staphylococcal enterotoxin B (SEB). A single immunoglobulin-like domain of the T-cell receptor (variable region, Vbeta) was subjected to multiple rounds of directed evolution using yeast display. Soluble forms of the engineered Vbeta proteins produced in Escherichia coli were effective inhibitors of SEB-mediated T-cell activation and completely neutralized the lethal activity of SEB in animal models. These Vbeta proteins represent an easily produced potential treatment for diseases mediated by bacterial superantigens.


Assuntos
Enterotoxinas/antagonistas & inibidores , Enterotoxinas/metabolismo , Fragmentos de Peptídeos/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Evolução Molecular Direcionada , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína/genética , Coelhos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Solubilidade
2.
J Mol Biol ; 371(1): 210-21, 2007 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-17560605

RESUMO

Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the alpha3-beta8 loop, a unique approximately 15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hVbeta5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG-TCR complexes, which results in the alpha3-beta8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR Vbeta domain specificity of SEK and other group V SAGs.


Assuntos
Proteínas de Bactérias/química , Enterotoxinas/química , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/química , Staphylococcus aureus/imunologia , Superantígenos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Cristalografia por Raios X , Enterotoxinas/imunologia , Humanos , Modelos Moleculares , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transdução de Sinais/fisiologia , Superantígenos/genética , Superantígenos/imunologia
3.
J Mol Biol ; 353(2): 308-21, 2005 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-16171815

RESUMO

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.


Assuntos
Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Superantígenos/imunologia , Superantígenos/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/química , Enterotoxinas/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Alinhamento de Sequência , Superantígenos/química , Ressonância de Plasmônio de Superfície
4.
Biochim Biophys Acta ; 1646(1-2): 49-56, 2003 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-12637011

RESUMO

We have carried out equilibrium studies of the effect of the amino acid residue difference in the primary structure of bovine cytochrome-c (b-cyt-c) and horse cyt-c (h-cyt-c) on the mechanism of their folding <--> unfolding processes at pH 6.0 and 25 degrees C. It has been observed that guanidinium chloride (GdmCl)-induced denaturation of b-cyt-c follows a two-state mechanism and that of h-cyt-c is not a two-state process. This conclusion is reached from the coincidence and non-coincidence of GdmCl-induced transition curves of bovine and horse proteins, respectively, monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. These measurements on h-cyt-c in the presence of GdmCl in the concentration range 0.75-2.0 M also suggest that the protein retains all the native far-UV CD but has slightly perturbed tertiary interaction. The intermediate in the presence of these low denaturant concentrations does not have the structural characteristics of a molten globule as judged by the 8-Anilino-1-napthalene sulfonic acid (ANS) binding and near-UV CD experiments. We have also carried out thermal denaturation studies of bovine and horse cyts-c in the presence of GdmCl monitored by absorbance at 405 nm and far-UV CD at 222 nm. The heat-induced denaturation measurements in the presence of the denaturant show (1) that denaturation of b-cyt-c is a two-state process and that of h-cyt-c does not follow a two-state mechanism, and (2) that the enthalpy change on denaturation of both proteins strongly depends on GdmCl concentration.


Assuntos
Grupo dos Citocromos c/química , Naftalenossulfonato de Anilina , Animais , Bovinos , Dicroísmo Circular , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Guanidina/farmacologia , Cavalos , Temperatura Alta , Concentração de Íons de Hidrogênio , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrofotometria , Temperatura
5.
J Infect Dis ; 198(3): 344-8, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18522504

RESUMO

Staphylococcus aureus secretes various toxins that act as superantigens by stimulating a large fraction of the host's T cells. Toxin binding to variable domains of T cell receptor beta chains (Vbeta) leads to massive release of inflammatory molecules and potentially to toxic shock syndrome (TSS). Previously, we generated soluble forms of different Vbeta domains with a high affinity for binding superantigens. However, a broader spectrum antagonist is required for the neutralization of multiple toxins. In the present study, we expressed Vbeta domains in tandem as a single-chain protein and neutralized the clinically important superantigens staphylococcal enterotoxin B and TSS toxin-1 with a single agent.


Assuntos
Antígenos de Bactérias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Receptores de Antígenos de Linfócitos T/genética
6.
Protein Expr Purif ; 54(1): 87-93, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17448676

RESUMO

Our goal is to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its receptor, the parathyroid hormone receptor type-1 (PTHR1). In order to study this interaction, we are aiming to obtain a three-dimensional structure of the PTH-PTHR1 bimolecular complex. Due to the very low expression levels of endogenous PTHR1, a recombinant form is required for structural analysis. However, the extreme hydrophobicity of the transmembrane regions of PTHR1 makes heterologous expression of PTHR1 difficult. Therefore, we sought to express the N-terminal extracellular domain (N-ECD) of PTHR1, a region that plays a pivotal role in ligand interaction. We expressed the N-ECD in both bacterial (Escherichia coli) and insect (Sf9) cells. The form produced in E. coli, a fusion-protein with thioredoxin, is soluble. However, removal of the fusion partner from a partially purified preparation results in dramatic loss of yield of the N-ECD. Expression in Sf9 cells, however, facilitates purification of a soluble form of the N-ECD. Isothermal calorimetry demonstrates that this N-ECD binds PTH-(1-34), albeit with lower affinity than the full-length receptor. This report describes the expression and purification of milligram quantities of the isolated N-ECD of PTHR1. The receptor fragment retains the ability to bind its cognate peptide ligand, an important pre-requisite for subsequent structural studies.


Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/biossíntese , Receptor Tipo 1 de Hormônio Paratireóideo/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Animais , Baculoviridae/genética , Escherichia coli/genética , Humanos , Insetos/citologia , Estrutura Terciária de Proteína/genética , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Proteínas Recombinantes/genética
7.
EMBO J ; 26(4): 1187-97, 2007 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-17268555

RESUMO

Superantigens (SAGs) bind simultaneously to major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, resulting in the massive release of inflammatory cytokines that can lead to toxic shock syndrome (TSS) and death. A major causative agent of TSS is toxic shock syndrome toxin-1 (TSST-1), which is unique relative to other bacterial SAGs owing to its structural divergence and its stringent TCR specificity. Here, we report the crystal structure of TSST-1 in complex with an affinity-matured variant of its wild-type TCR ligand, human T-cell receptor beta chain variable domain 2.1. From this structure and a model of the wild-type complex, we show that TSST-1 engages TCR ligands in a markedly different way than do other SAGs. We provide a structural basis for the high TCR specificity of TSST-1 and present a model of the TSST-1-dependent MHC-SAG-TCR T-cell signaling complex that is structurally and energetically unique relative to those formed by other SAGs. Our data also suggest that protein plasticity plays an exceptionally significant role in this affinity maturation process that results in more than a 3000-fold increase in affinity.


Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Epitopos/genética , Modelos Moleculares , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais/imunologia , Superantígenos/química , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Toxinas Bacterianas/metabolismo , Cristalografia , Enterotoxinas/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica , Superantígenos/metabolismo , Ressonância de Plasmônio de Superfície
8.
J Exp Med ; 204(12): 2813-24, 2007 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-18025130

RESUMO

Viral mutational escape can reduce or abrogate recognition by the T cell receptor (TCR) of virus-specific CD8+ T cells. However, very little is known about the impact of cytotoxic T lymphocyte (CTL) epitope mutations on interactions between peptide-major histocompatibility complex (MHC) class I complexes and MHC class I receptors expressed on other cell types. Here, we analyzed a variant of the immunodominant human leukocyte antigen (HLA)-B2705-restricted HIV-1 Gag KK10 epitope (KRWIILGLNK) with an L to M amino acid substitution at position 6 (L6M), which arises as a CTL escape variant after primary infection but is sufficiently immunogenic to elicit a secondary, de novo HIV-1-specific CD8+ T cell response with an alternative TCR repertoire in chronic infection. In addition to altering recognition by HIV-1-specific CD8+ T cells, the HLA-B2705-KK10 L6M complex also exhibits substantially increased binding to the immunoglobulin-like transcript (ILT) receptor 4, an inhibitory MHC class I-specific receptor expressed on myelomonocytic cells. Binding of the B2705-KK10 L6M complex to ILT4 leads to a tolerogenic phenotype of myelomonocytic cells with lower surface expression of dendritic cell (DC) maturation markers and co-stimulatory molecules. These data suggest a link between CTL-driven mutational escape, altered recognition by innate MHC class I receptors on myelomonocytic cells, and functional impairment of DCs, and thus provide important new insight into biological consequences of viral sequence diversification.


Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/imunologia , HIV/imunologia , Monócitos/imunologia , Células Mieloides/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Síndrome da Imunodeficiência Adquirida/genética , HIV/genética , Antígeno HLA-B27/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Monócitos/virologia , Mutação , Células Mieloides/virologia , Receptores de Antígenos de Linfócitos T/genética
9.
Biochemistry ; 45(14): 4695-702, 2006 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-16584204

RESUMO

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state <--> MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.


Assuntos
Citocromos c/química , Compostos de Lítio/farmacologia , Percloratos/farmacologia , Conformação Proteica/efeitos dos fármacos , Animais , Bovinos , Dicroísmo Circular , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Espectrofotometria , Termodinâmica
10.
J Immunol ; 177(12): 8595-603, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17142758

RESUMO

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (Vbetas) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vbeta2(+) T cells. Herein, we have analyzed the SpeC-Vbeta2.1 interaction by mutating all SpeC residues that make contact with Vbeta2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vbeta2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with Vbeta2.1. Three of these residues (Tyr(15), Phe(75), and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in Vbeta2 and Vbeta4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vbeta2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vbeta domain subfamily.


Assuntos
Proteínas de Bactérias/metabolismo , Reações Cruzadas/imunologia , Exotoxinas/metabolismo , Mapeamento de Interação de Proteínas , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Superantígenos/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Alelos , Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Linhagem Celular , Epitopos , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/imunologia , Humanos , Células Jurkat , Mutagênese Sítio-Dirigida , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Ressonância de Plasmônio de Superfície
11.
Proc Natl Acad Sci U S A ; 103(26): 9867-72, 2006 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-16788072

RESUMO

Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by >20 A. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single beta-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.


Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Superantígenos/química , Humanos , Mutagênese , Mutação , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Ressonância de Plasmônio de Superfície
12.
Biochemistry ; 42(6): 1684-95, 2003 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-12578383

RESUMO

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.


Assuntos
Grupo dos Citocromos c/química , Desnaturação Proteica , Dobramento de Proteína , Sais/química , Termodinâmica , Animais , Cloreto de Cálcio/química , Bovinos , Dicroísmo Circular , Estabilidade Enzimática , Cavalos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Cloreto de Lítio/química , Modelos Químicos , Conformação Proteica , Estrutura Secundária de Proteína , Temperatura
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