RESUMO
Human T cell antigen receptors (TCRs) pair in millions of combinations to create complex and unique T cell repertoires for each person. Through the use of tetramers to analyze TCRs reactive to the antigen-presenting molecule CD1b, we detected T cells with highly stereotyped TCR α-chains present among genetically unrelated patients with tuberculosis. The germline-encoded, mycolyl lipid-reactive (GEM) TCRs had an α-chain bearing the variable (V) region TRAV1-2 rearranged to the joining (J) region TRAJ9 with few nontemplated (N)-region additions. Analysis of TCRs by high-throughput sequencing, binding and crystallography showed linkage of TCRα sequence motifs to high-affinity recognition of antigen. Thus, the CD1-reactive TCR repertoire is composed of at least two compartments: high-affinity GEM TCRs, and more-diverse TCRs with low affinity for CD1b-lipid complexes. We found high interdonor conservation of TCRs that probably resulted from selection by a nonpolymorphic antigen-presenting molecule and an immunodominant antigen.
Assuntos
Antígenos CD1/imunologia , Infecções por Mycobacterium/imunologia , Mycobacterium/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Sequência de Bases , Células Clonais , Cristalografia por Raios X , Citometria de Fluxo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Infecções por Mycobacterium/microbiologia , RNA/química , RNA/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Subpopulações de Linfócitos T/citologia , Linfócitos T/citologiaRESUMO
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
Current views emphasize TCR diversity as a key feature that differentiates the group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 systems. Whereas TCR sequence motifs define CD1d-reactive NKT cells, the available data do not allow a TCR-based organization of the group 1 CD1 repertoire. The observed TCR diversity might result from donor-to-donor differences in TCR repertoire, as seen for MHC-restricted T cells. Alternatively, diversity might result from differing CD1 isoforms, Ags, and methods used to identify TCRs. Using CD1b tetramers to isolate clones recognizing the same glycolipid, we identified a previously unknown pattern of V gene usage (TRAV17, TRBV4-1) among unrelated human subjects. These TCRs are distinct from those present on NKT cells and germline-encoded mycolyl lipid-reactive T cells. Instead, they resemble the TCR of LDN5, one of the first known CD1b-reactive clones that was previously thought to illustrate the diversity of the TCR repertoire. Interdonor TCR conservation was observed in vitro and ex vivo, identifying LDN5-like T cells as a distinct T cell type. These data support TCR-based organization of the CD1b repertoire, which consists of at least two compartments that differ in TCR sequence motifs, affinity, and coreceptor expression.
Assuntos
Motivos de Aminoácidos/imunologia , Receptores de Antígenos de Linfócitos T/química , Subpopulações de Linfócitos T/química , Antígenos CD1/imunologia , Sequência de Bases , Sequência Conservada/imunologia , Citometria de Fluxo , Glicolipídeos/imunologia , Humanos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologiaRESUMO
Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 µM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.
Assuntos
Antígenos CD1/metabolismo , Lipopeptídeos/metabolismo , Oxazóis/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linhagem Celular , Células HEK293 , Humanos , Lipopeptídeos/imunologia , Ativação Linfocitária/imunologia , Oxazóis/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Tuberculose/imunologiaRESUMO
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
Combinations of LIM homeodomain proteins form a transcriptional "LIM code" to direct the specification of neural cell types. Two paralogous pairs of LIM homeodomain proteins, LIM homeobox protein 3/4 (Lhx3/Lhx4) and Islet-1/2 (Isl1/Isl2), are expressed in developing ventral motor neurons. Lhx3 and Isl1 interact within a well characterized transcriptional complex that triggers motor neuron development, but it was not known whether Lhx4 and Isl2 could participate in equivalent complexes. We have identified an Lhx3-binding domain (LBD) in Isl2 based on sequence homology with the Isl1(LBD) and show that both Isl2(LBD) and Isl1(LBD) can bind each of Lhx3 and Lhx4. X-ray crystal- and small-angle x-ray scattering-derived solution structures of an Lhx4·Isl2 complex exhibit many similarities with that of Lhx3·Isl1; however, structural differences supported by mutagenic studies reveal differences in the mechanisms of binding. Differences in binding have implications for the mode of exchange of protein partners in transcriptional complexes and indicate a divergence in functions of Lhx3/4 and Isl1/2. The formation of weaker Lhx·Isl complexes would likely be masked by the availability of the other Lhx·Isl complexes in postmitotic motor neurons.
Assuntos
Fatores de Transcrição/metabolismo , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Homeodomínio LIM/química , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Mutagênese , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1(LBD)). Although the LIM interaction domain of Ldb1 (Ldb1(LID)) and Isl1(LBD) share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1(LBD) mimics Ldb1(LID). These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Domínio LIM , Proteínas com Homeodomínio LIM , Modelos Moleculares , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Termodinâmica , Fatores de Transcrição , Técnicas do Sistema de Duplo-HíbridoRESUMO
LMO (LIM-only) and LIM-HD (LIM-homeodomain) proteins form a family of proteins that is required for myriad developmental processes and which can contribute to diseases such as T-cell leukaemia and breast cancer. The four LMO and 12 LIM-HD proteins in mammals are expressed in a combinatorial manner in many cell types, forming a transcriptional 'LIM code'. The proteins all contain a pair of closely spaced LIM domains near their N-termini that mediate protein-protein interactions, including binding to the approximately 30-residue LID (LIM interaction domain) of the essential co-factor protein Ldb1 (LIM domain-binding protein 1). In an attempt to understand the molecular mechanisms behind the LIM code, we have determined the molecular basis of binding of LMO and LIM-HD proteins for Ldb1(LID) through a series of structural, mutagenic and biophysical studies. These studies provide an explanation for why Ldb1 binds the LIM domains of the LMO/LIM-HD family, but not LIM domains from other proteins. The LMO/LIM-HD family exhibit a range of affinities for Ldb1, which influences the formation of specific functional complexes within cells. We have also identified an additional LIM interaction domain in one of the LIM-HD proteins, Isl1. Despite low sequence similarity to Ldb1(LID), this domain binds another LIM-HD protein, Lhx3, in an identical manner to Ldb1(LID). Through our and other studies, it is emerging that the multiple layers of competitive binding involving LMO and LIM-HD proteins and their partner proteins contribute significantly to cell fate specification and development.
Assuntos
Ligação Competitiva , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Estrutura Terciária de ProteínaRESUMO
A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Lhx3 tethered to a peptide region of Isl1 has been engineered, purified and crystallized. The monoclinic crystals belong to space group C2, with unit-cell parameters a = 119, b = 62.2, c = 51.9 A, beta = 91.6 degrees , and diffract to 2.05 A resolution.
Assuntos
Proteínas de Homeodomínio/química , Cristalização , Vetores Genéticos/síntese química , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/síntese química , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Engenharia de Proteínas , Fatores de Transcrição , Dedos de Zinco/genéticaRESUMO
LIM-Homeodomain (LIM-HD) transcription factors are highly conserved in animals where they are thought to act in a transcriptional 'LIM code' that specifies cell types, particularly in the central nervous system. In chick and mammals the interaction between two LIM-HD proteins, LHX3 and Islet1 (ISL1), is essential for the development of motor neurons. Using yeast two-hybrid analysis we showed that the Caenorhabditis elegans orthologs of LHX3 and ISL1, CEH-14 and LIM-7 can physically interact. Structural characterisation of a complex comprising the LIM domains from CEH-14 and a LIM-interaction domain from LIM-7 showed that these nematode proteins assemble to form a structure that closely resembles that of their vertebrate counterparts. However, mutagenic analysis across the interface indicates some differences in the mechanisms of binding. We also demonstrate, using fluorescent reporter constructs, that the two C. elegans proteins are co-expressed in a small subset of neurons. These data show that the propensity for LHX3 and Islet proteins to interact is conserved from C. elegans to mammals, raising the possibility that orthologous cell specific LIM-HD-containing transcription factor complexes play similar roles in the development of neuronal cells across diverse species.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação , Caenorhabditis elegans/genética , Sequência Conservada , Evolução Molecular , Regulação da Expressão Gênica , Proteínas com Homeodomínio LIM/química , Proteínas com Homeodomínio LIM/genética , Modelos Moleculares , Família Multigênica , Complexos Multiproteicos , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Soluções , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
CD1 proteins present microbial lipids to T cells. Germline-encoded mycolyl lipid-reactive (GEM) T cells with conserved αß T cell receptors (TCRs) recognize CD1b presenting mycobacterial mycolates. As the molecular basis underpinning TCR recognition of CD1b remains unknown, here we determine the structure of a GEM TCR bound to CD1b presenting glucose-6-O-monomycolate (GMM). The GEM TCR docks centrally above CD1b, whereby the conserved TCR α-chain extensively contacts CD1b and GMM. Through mutagenesis and study of T cells from tuberculosis patients, we identify a consensus CD1b footprint of TCRs present among GEM T cells. Using both the TCR α- and ß-chains as tweezers to surround and grip the glucose moiety of GMM, GEM TCRs create a highly specific mechanism for recognizing this mycobacterial glycolipid.
Assuntos
Antígenos CD1/metabolismo , Glicolipídeos/imunologia , Tuberculose Latente/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Mycobacterium phlei , Conformação Proteica , Rhodococcus equiRESUMO
The T-cell antigen receptor is a heterodimeric αß protein (TCR) expressed on the surface of T-lymphocytes, with each chain of the TCR comprising three complementarity-determining regions (CDRs) that collectively form the antigen-binding site. Unlike antibodies, which are closely related proteins that recognize intact protein antigens, TCRs classically bind, via their CDR loops, to peptides (p) that are presented by molecules of the major histocompatibility complex (MHC). This TCR-pMHC interaction is crucially important in cell-mediated immunity, with the specificity in the cellular immune response being attributable to MHC polymorphism, an extensive TCR repertoire and a variable peptide cargo. The ensuing structural and biophysical studies within the TCR-pMHC axis have been highly informative in understanding the fundamental events that underpin protective immunity and dysfunctional T-cell responses that occur during autoimmunity. In addition, TCRs can recognize the CD1 family, a family of MHC-related molecules that instead of presenting peptides are ideally suited to bind lipid-based antigens. Structural studies within the CD1-lipid antigen system are beginning to inform us how lipid antigens are specifically presented by CD1, and how such CD1-lipid antigen complexes are recognized by the TCR. Moreover, it has recently been shown that certain TCRs can bind to vitamin B based metabolites that are bound to an MHC-like molecule termed MR1. Thus, TCRs can recognize peptides, lipids, and small molecule metabolites, and here we review the basic principles underpinning this versatile and fascinating receptor recognition system that is vital to a host's survival.
Assuntos
Peptídeos/classificação , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Regiões Determinantes de Complementaridade/metabolismo , Humanos , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Modelos Moleculares , Peptídeos/química , Polimorfismo GenéticoRESUMO
Lhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and Ldb1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an Ldb1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in Ldb1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both Ldb1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas com Domínio LIM/química , Proteínas com Homeodomínio LIM/química , Mutação , Fatores de Transcrição/química , Motivos de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mucosal-associated invariant T (MAIT) cells express a semiinvariant αß T cell receptor (TCR) that binds MHC class I-like molecule (MR1). However, the molecular basis for MAIT TCR recognition by MR1 is unknown. In this study, we present the crystal structure of a human Vα7.2Jα33-Vß2 MAIT TCR. Mutagenesis revealed highly conserved requirements for the MAIT TCR-MR1 interaction across different human MAIT TCRs stimulated by distinct microbial sources. Individual residues within the MAIT TCR ß chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR α chain controlled specificity through a small number of residues, which are conserved across species and located within the Vα-Jα regions. Mutagenesis of MR1 showed that only two residues, which were centrally positioned and on opposing sides of the antigen-binding cleft of MR1, were essential for MAIT cell activation. The mutagenesis data are consistent with a centrally located MAIT TCR-MR1 docking that was dominated by the α chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR-CD1d-antigen interaction, in which both the α and ß chain of the NKT TCR is required for ligation above the F'-pocket of CD1d.