RESUMO
Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes.
Assuntos
Butirofilinas , Receptores de Antígenos de Linfócitos T gama-delta , Butirofilinas/metabolismo , Butirofilinas/imunologia , Butirofilinas/química , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Humanos , Ligação Proteica , Multimerização Proteica , Antígenos CD/metabolismo , Antígenos CD/imunologia , Antígenos CD/química , Linfócitos T/imunologia , Linfócitos T/metabolismo , Cristalografia por Raios X , Ativação Linfocitária/imunologia , Modelos Moleculares , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismoRESUMO
The T cell repertoire comprises αß and γδ T cell lineages. Although it is established how αß T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for γδ TCRs. We describe a population of human Vδ1(+) γδ T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a γδ TCR binds CD1d-α-galactosylceramide (α-GalCer). The γδ TCR docked orthogonally, over the A' pocket of CD1d, in which the Vδ1-chain, and in particular the germ line-encoded CDR1δ loop, dominated interactions with CD1d. The TCR γ-chain sat peripherally to the interface, with the CDR3γ loop representing the principal determinant for α-GalCer specificity. Accordingly, we provide insight into how a γδ TCR binds specifically to a lipid-loaded antigen-presenting molecule.
Assuntos
Antígenos CD1d/química , Galactosilceramidas/química , Simulação de Acoplamento Molecular , Receptores de Antígenos de Linfócitos T gama-delta/química , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Antígenos CD1d/imunologia , Sítios de Ligação , Bases de Dados de Proteínas , Galactosilceramidas/imunologia , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/citologiaRESUMO
Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRß chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.
Assuntos
Antígenos CD1d/imunologia , Células Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Sulfoglicoesfingolipídeos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos CD1d/química , Cristalização , Células Matadoras Naturais/química , Ativação Linfocitária , Camundongos , Reação em Cadeia da Polimerase , Estrutura Quaternária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sulfoglicoesfingolipídeos/química , Ressonância de Plasmônio de Superfície , Subpopulações de Linfócitos T/químicaRESUMO
The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct ß-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated ß-linked agonists ß-galactosylceramide (ß-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the ß-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the ß-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to ß-linked antigens.
Assuntos
Antígenos CD1d/imunologia , Autoimunidade , Galactosilceramidas/imunologia , Globosídeos/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Triexosilceramidas/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD1d/química , Antígenos CD1d/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citometria de Fluxo , Galactosilceramidas/química , Galactosilceramidas/metabolismo , Globosídeos/química , Globosídeos/metabolismo , Humanos , Hibridomas , Cinética , Camundongos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/metabolismo , Ligação Proteica/imunologia , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Triexosilceramidas/química , Triexosilceramidas/metabolismoRESUMO
Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.
Assuntos
Galactosilceramidas/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Antígenos CD1d/imunologia , Linhagem Celular , Galactosilceramidas/farmacologia , Glucuronatos/imunologia , Humanos , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.
Assuntos
Receptores da Família Eph/química , Receptores da Família Eph/metabolismo , Transdução de Sinais/genética , Motivo Estéril alfa/genética , Domínios de Homologia de src/genética , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Inibidores de Proteínas Quinases/metabolismo , Receptores da Família Eph/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera/citologia , Tirosina/metabolismoRESUMO
The antigen receptor for natural killer T cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3 Å resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3ß loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.
Assuntos
Antígenos CD1d/imunologia , Autoantígenos , Células T Matadoras Naturais/imunologia , Animais , Cristalografia por Raios X , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Complexos Multiproteicos , Receptores de Células Matadoras Naturais/imunologiaRESUMO
Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.
Assuntos
Antígenos CD1d/imunologia , Epitopos , Células T Matadoras Naturais/imunologia , Animais , Sequência de Carboidratos , Linhagem Celular , Proliferação de Células , Glicolipídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Células T Matadoras Naturais/citologia , Receptores de Células Matadoras Naturais/imunologiaRESUMO
T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Ativação Linfocitária/imunologia , Redes e Vias Metabólicas , Pirimidinas/metabolismo , Riboflavina/metabolismo , Subpopulações de Linfócitos T/imunologia , Amino Açúcares/química , Amino Açúcares/imunologia , Amino Açúcares/metabolismo , Apresentação de Antígeno/imunologia , Antígenos de Bactérias/química , Glioxal/química , Glioxal/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Inata/imunologia , Imunidade nas Mucosas/imunologia , Ligantes , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Conformação Molecular , Mucosa/imunologia , Pirimidinas/química , Pirimidinas/imunologia , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo , Riboflavina/biossíntese , Riboflavina/imunologia , Bases de Schiff/química , Subpopulações de Linfócitos T/citologia , Uracila/análogos & derivados , Uracila/química , Uracila/imunologia , Uracila/metabolismo , Complexo Vitamínico B/imunologia , Complexo Vitamínico B/metabolismoRESUMO
Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.
Assuntos
Antígenos CD1d/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Antígenos CD1d/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timo/imunologiaRESUMO
The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.
Assuntos
Antígenos CD1d/imunologia , Galactosilceramidas/imunologia , Células T Matadoras Naturais/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Antígenos CD1d/química , Clonagem Molecular , Cristalização , Galactosilceramidas/química , Humanos , Camundongos , Mutagênese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Antigen-presenting molecules, encoded by the major histocompatibility complex (MHC) and CD1 family, bind peptide- and lipid-based antigens, respectively, for recognition by T cells. Mucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T cells in humans that are activated by an antigen(s) bound to the MHC class I-like molecule MR1. Although the identity of MR1-restricted antigen(s) is unknown, it is present in numerous bacteria and yeast. Here we show that the structure and chemistry within the antigen-binding cleft of MR1 is distinct from the MHC and CD1 families. MR1 is ideally suited to bind ligands originating from vitamin metabolites. The structure of MR1 in complex with 6-formyl pterin, a folic acid (vitamin B9) metabolite, shows the pterin ring sequestered within MR1. Furthermore, we characterize related MR1-restricted vitamin derivatives, originating from the bacterial riboflavin (vitamin B2) biosynthetic pathway, which specifically and potently activate MAIT cells. Accordingly, we show that metabolites of vitamin B represent a class of antigen that are presented by MR1 for MAIT-cell immunosurveillance. As many vitamin biosynthetic pathways are unique to bacteria and yeast, our data suggest that MAIT cells use these metabolites to detect microbial infection.
Assuntos
Ácido Fólico/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Pterinas/química , Pterinas/imunologia , Linfócitos T/imunologia , Apresentação de Antígeno , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Ácido Fólico/química , Ácido Fólico/imunologia , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vigilância Imunológica/imunologia , Células Jurkat , Ligantes , Ativação Linfocitária , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Redobramento de Proteína/efeitos dos fármacos , Pterinas/metabolismo , Pterinas/farmacologia , Salmonella/imunologia , Salmonella/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Eletricidade Estática , Microglobulina beta-2/imunologia , Microglobulina beta-2/metabolismoRESUMO
Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d-presented self-antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3-dependent human iNKT-cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human-mouse cross-species analysis of iNKT-cell activation by iGb3-CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3-hCD1d was unable to support cognate interactions with the iNKT-cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine-to-tryptophan modification within the α2-helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3-hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT-cell biology.
Assuntos
Antígenos CD1d/imunologia , Globosídeos/imunologia , Células T Matadoras Naturais/imunologia , Triexosilceramidas/imunologia , Aminoácidos , Animais , Apresentação de Antígeno , Antígenos CD1d/química , Antígenos CD1d/metabolismo , Globosídeos/metabolismo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade da Espécie , Triexosilceramidas/metabolismoRESUMO
alphabeta T cell receptors (TCRs) have traditionally been viewed as receptors for peptide antigens presented by either Major Histocompatibility Complex (MHC) class I (for CD8 T cells) or MHC class II (for CD4 T cells) antigen-presenting molecules. However, it is now clear that some T cell lineages express TCRs that are specialized for recognition of lipid-based antigens presented by the MHC class I-like CD1 family. Recently, the molecular basis for the TCR recognition of glycolipid antigens presented by CD1d has revealed an evolutionarily conserved-docking mode that is distinct from that of peptide-based recognition. T cells carrying these receptors follow a unique developmental pathway that results not only in unconventional antigen specificity, but also seemingly exaggerated functional capabilities, which makes these cells and their antigens highly attractive targets for immunotherapeutic manipulation.
Assuntos
Antígenos CD1d/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antígenos/imunologia , Humanos , Imunidade Inata , Lipídeos/imunologia , Ligação Proteica , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR α-chain is invariant, NKT TCR Vß exhibits greater diversity, with one (Vß11) and three (Vß8, Vß7, and Vß2) Vß chains in humans and mice, respectively. With the exception of the Vß2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2ß that are critical for CD1d binding. Thus, how Vß2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2ß-encoded tyrosine residues, we show that the Vß2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the Vß8.2 and Vß7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR ß-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the Vß2 NKT TCR and the Vß8.2 and Vß7 NKT TCRs, with the Vß2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the Vß2 NKT TCR-CD1d-αGalCer complex, the CDR2ß loop mediated fewer contacts with CD1d, whereas the CDR1ß and CDR3ß loops contacted CD1d to a much greater extent compared with most Vß11, Vß8.2, and Vß7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR ß-chain of the Vß2 NKT TCR that enables CD1d-Ag ligation.
Assuntos
Antígenos CD1d/imunologia , Glicolipídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Clonagem Molecular , Epitopos/genética , Epitopos/imunologia , Citometria de Fluxo , Glicolipídeos/metabolismo , Camundongos , Mutagênese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4',4â³-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4'-OH position in comparison with the 3'-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4'-OH > 3'-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.
Assuntos
Antígenos CD1d/metabolismo , Células T Matadoras Naturais/imunologia , Receptores de Antígenos/metabolismo , Motivos de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos/química , Cristalografia por Raios X/métodos , Citometria de Fluxo/métodos , Glicolipídeos/química , Humanos , Leucócitos Mononucleares/citologia , Lipídeos/química , Camundongos , Modelos Moleculares , Conformação Molecular , Células T Matadoras Naturais/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.
Assuntos
Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adaptação Biológica/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos CD1d/química , Antígenos Glicosídicos Associados a Tumores/imunologia , Células Cultivadas , Galactosilceramidas/química , Galactosilceramidas/imunologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Relação Estrutura-Atividade , Especificidade do Receptor de Antígeno de Linfócitos T/imunologiaRESUMO
NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vß8.2 and Vß7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is â¼10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vß8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.
Assuntos
Antígenos CD1d/metabolismo , Galactosilceramidas/metabolismo , Células T Matadoras Naturais/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Antígenos CD1d/imunologia , Configuração de Carboidratos , Células Cultivadas , Cristalografia por Raios X , Galactosilceramidas/imunologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.
Assuntos
Proteínas 14-3-3 , Proteínas do Citoesqueleto , Transdução de Sinais , Movimento Celular , Proliferação de Células , Transdução de Sinais/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas 14-3-3/metabolismoRESUMO
The PEAK family of pseudokinases, which comprises PEAK1, PEAK2 and PEAK3, are newly identified scaffolds that dynamically assemble oncogenic signaling pathways known to contribute to the development of several aggressive cancers. A striking feature of this unique family of pseudokinase scaffolds is their large multi-domain structure, which allows them to achieve protein complex assemblies through their structural plasticity and functional versatility. Recent structural advances have begun to reveal the critical regulatory elements that control their function. Specifically, the dimer-dependent scaffolding activity of PEAK pseudokinases is emerging as a critical mechanism for their signaling function, in addition to their ability to hetero-associate to form higher-order regulatory networks to diversify and amplify their signaling output. Here, we present a suite of techniques that enable the efficient expression and purification of PEAK proteins for functional characterization.