RESUMO
T cells in jawed vertebrates comprise two lineages, αß T-cells and γδ T-cells, defined by the antigen receptors they express, i.e., αß and γδ T-cell receptors (TCRs), respectively. The two lineages have different immunological roles, requiring γδ TCRs to recognize more structurally-diverse ligands1. Nevertheless, the receptors use shared CD3 subunits to initiate signaling. Whereas the structural organization of αß TCRs is understood2,3, the architecture of γδ TCRs is unknown. Here, we used cryogenic electron microscopy to determine the structure of a fully-assembled, MR1-reactive human Vδ3Vγ8 TCR/CD3δγε2ζ2 complex bound by anti-CD3ε antibody Fab fragments4,5. The arrangement of CD3 subunits in γδ and αß TCRs is conserved and, although the transmembrane α-helices of the TCR-γδ and -αß subunits differ markedly in sequence, the packing of the eight transmembrane-helix bundles is similar6. However, in contrast to the apparently rigid αß TCR2,3,6, the γδ TCR exhibits considerable conformational heterogeneity, owing to the ligand-binding TCR-γδ subunits being tethered to the CD3 subunits by their transmembrane regions only. Reducing this conformational heterogeneity by transferring the Vδ3Vγ8 TCR variable domains to an αß TCR enhanced receptor signaling, suggesting that γδ TCR organization reflects a compromise between efficient signaling and the ability to engage structurally-diverse ligands. Our findings reveal the remarkable structural plasticity of the TCR on evolutionary timescales, and recast it as a highly versatile receptor capable of initiating signaling as either a rigid or flexible structure.
RESUMO
Unlike conventional αß T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1+ and Vδ2+ γδ TCR-mediated ligand recognition, the mode of Vδ3+ TCR ligand engagement is unknown. MHC class I-related protein, MR1, presents vitamin B metabolites to αß T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2- γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3+ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3+ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Linfócitos Intraepiteliais/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Adulto , Apresentação de Antígeno , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Humanos , Linfócitos Intraepiteliais/fisiologia , Ligantes , Masculino , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/fisiologia , Células T Invariantes Associadas à Mucosa/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/fisiologiaRESUMO
In this article, we discuss recent advances into the structural analyses of immunoglobulin M complexes, which are enabling comprehensive characterization of these enigmatic antibodies, to reveal central tenets of immunoglobulin M immunobiology and inform their immunotherapeutic use.
Assuntos
Biologia , Imunoglobulina M , Microscopia CrioeletrônicaRESUMO
DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.
Assuntos
Adjuvantes Imunológicos , Oligodesoxirribonucleotídeos , Animais , Células Dendríticas , Interleucina-12 , Fígado , CamundongosRESUMO
γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αß TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids.
Assuntos
Antígenos CD1/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Apresentação de Antígeno , Antígenos CD1/imunologia , Cristalografia por Raios X/métodos , Humanos , Linfócitos Intraepiteliais/fisiologia , Lipídeos/imunologia , Ativação Linfocitária/imunologia , Modelos Moleculares , Monócitos/metabolismo , Linfócitos T/imunologiaRESUMO
The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of "latching" N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9's role within SARS-CoV-2 replication.
Assuntos
COVID-19/virologia , Nucleotídeos de Pirimidina/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , RNA/metabolismo , SARS-CoV-2/fisiologia , Replicação ViralRESUMO
The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Diterpenos do Tipo Caurano/farmacologia , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Sítios de Ligação/efeitos dos fármacos , Produtos Biológicos/química , Produtos Biológicos/farmacologia , COVID-19/metabolismo , COVID-19/virologia , Chlorocebus aethiops , Diterpenos do Tipo Caurano/química , Humanos , Simulação de Acoplamento Molecular , Proteínas de Ligação a RNA/química , SARS-CoV-2/química , SARS-CoV-2/fisiologia , Células Vero , Proteínas não Estruturais Virais/químicaRESUMO
DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine-guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine-guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Microscopia Crioeletrônica/métodos , Fibronectinas/metabolismo , Lectinas Tipo C/metabolismo , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Humanos , Lectinas Tipo C/química , Ligantes , Conformação ProteicaRESUMO
The γδ T cell immune cell lineage has remained relatively enigmatic and under-characterised since their identification. Conversely, the insights we have, highlight their central importance in diverse immunological roles and homeostasis. Thus, γδ T cells are considered as potentially a new translational tool in the design of new therapeutics for cancer and infectious disease. Here we review our current understanding of γδ T cell biology viewed through a structural lens centred on the how the γδ T cell receptor mediates ligand recognition. We discuss the limited knowledge of antigens, the structural basis of such reactivities and discuss the emerging trends of γδ T cell reactivity and implications for γδ T cell biology.
Assuntos
Imunidade Celular , Linfócitos Intraepiteliais/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Animais , Antígenos/imunologia , Linhagem da Célula/imunologia , Doenças Transmissíveis/imunologia , Homeostase/imunologia , Humanos , Ligantes , Neoplasias/imunologiaRESUMO
The race to identify a successful treatment for COVID19 will be defined by fundamental research into the replication cycle of the SARS-CoV-2 virus. This has identified five distinct stages from which numerous vaccination and clinical trials have emerged alongside an innumerable number of drug discovery studies currently in development for disease intervention. Informing every step of the viral replication cycle has been an unprecedented 'call-to-arms' by the global structural biology community. Of the 20 main SARS-CoV-2 proteins, 13 have been resolved structurally for SARS-CoV-2 with most having a related SARS-CoV and MERS-CoV structural homologue totalling some 300 structures currently available in public repositories. Herein, we review the contribution of structural studies to our understanding of the virus and their role in structure-based development of therapeutics.
Assuntos
Antivirais/química , Antivirais/uso terapêutico , COVID-19/terapia , Descoberta de Drogas/métodos , SARS-CoV-2 , Antivirais/síntese química , COVID-19/imunologia , Desenvolvimento de Medicamentos/métodos , Genoma Viral , Humanos , Modelos Moleculares , Elementos Estruturais de Proteínas , SARS-CoV-2/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Relação Estrutura-Atividade , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia , Tratamento Farmacológico da COVID-19RESUMO
T cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed on the surface of natural killer (NK) cells. TIGIT recognizes nectin and nectin-like adhesion molecules and thus plays a critical role in the innate immune response to malignant transformation. Although the TIGIT nectin-like protein-5 (necl-5) interaction is well understood, how TIGIT engages nectin-2, a receptor that is broadly over-expressed in breast and ovarian cancer, remains unknown. Here, we show that TIGIT bound to the immunoglobulin domain of nectin-2 that is most distal from the membrane with an affinity of 6 µm, which was moderately lower than the affinity observed for the TIGIT/necl-5 interaction (3.2 µm). The TIGIT/nectin-2 binding disrupted pre-assembled nectin-2 oligomers, suggesting that receptor-ligand and ligand-ligand associations are mutually exclusive events. Indeed, the crystal structure of TIGIT bound to the first immunoglobulin domain of nectin-2 indicated that the receptor and ligand dock using the same molecular surface and a conserved "lock and key" binding motifs previously observed to mediate nectin/nectin homotypic interactions as well as TIGIT/necl-5 recognition. Using a mutagenesis approach, we dissected the energetic basis for the TIGIT/nectin-2 interaction and revealed that an "aromatic key" of nectin-2 is critical for this interaction, whereas variations in the lock were tolerated. Moreover, we found that the C-C' loop of the ligand dictates the TIGIT binding hierarchy. Altogether, these findings broaden our understanding of nectin/nectin receptor interactions and have implications for better understanding the molecular basis for autoimmune disease and cancer.
Assuntos
Moléculas de Adesão Celular/química , Receptores Imunológicos/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cristalografia por Raios X , Humanos , Mutagênese , Nectinas , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
The pentatricopeptide repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an α-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts ZEA MAYS: PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, ATPH: and PSAJ, HAS BEEN DEMONSTRATED TO FOLLOW: a recognition code where the identity of two amino acids per repeat determines the base-specificity. A recently solved ZmPPR10: PSAJ: complex crystal structure suggested a homodimeric complex with considerably fewer sequence-specific protein-RNA contacts than inferred PREVIOUSLY: Here we describe the solution structure of the ZmPPR10: ATPH: complex using size-exclusion chromatography-coupled synchrotron small-angle X-ray scattering (SEC-SY-SAXS). Our results support prior evidence that PPR10 binds RNA as a monomer, and that it does so in a manner that is commensurate with a canonical and predictable RNA-binding mode across much of the RNA-protein interface.
Assuntos
Proteínas de Plantas/química , RNA de Plantas/metabolismo , Zea mays/metabolismo , Dicroísmo Circular , Modelos Moleculares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espalhamento de Radiação , Zea mays/genéticaRESUMO
SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism.
Assuntos
Regulação da Expressão Gênica/fisiologia , Polímeros/química , Proteínas de Ligação a RNA/química , Western Blotting , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Microscopia Eletrônica de Transmissão , Fator de Processamento Associado a PTB , Conformação Proteica , Proteínas de Ligação a RNA/fisiologiaRESUMO
Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2â Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Ligação a RNA/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Arabidopsis/síntese química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Ligação a RNA/síntese química , Alinhamento de SequênciaRESUMO
CD1a is a monomorphic antigen-presenting molecule on dendritic cells that presents lipids to αß T cells. Whether CD1a represents a ligand for other immune receptors remains unknown. Here we use CD1a tetramers to show that CD1a is a ligand for Vδ1+ γδ T cells. Functional studies suggest that two γδ T cell receptors (TCRs) bound CD1a in a lipid-independent manner. The crystal structures of three Vγ4Vδ1 TCR-CD1a-lipid complexes reveal that the γδ TCR binds at the extreme far side and parallel to the long axis of the ß-sheet floor of CD1a's antigen-binding cleft. Here, the γδ TCR co-recognises the CD1a heavy chain and ß2 microglobulin in a manner that is distinct from all other previously observed γδ TCR docking modalities. The 'sideways' and lipid antigen independent mode of autoreactive CD1a recognition induces TCR clustering on the cell surface and proximal T cell signalling as measured by CD3ζ phosphorylation. In contrast with the 'end to end' binding of αß TCRs that typically contact carried antigens, autoreactive γδ TCRs support geometrically diverse approaches to CD1a, as well as antigen independent recognition.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T , Antígenos , Ligantes , Lipídeos/análise , Receptores de Antígenos de Linfócitos T gama-delta/metabolismoRESUMO
Many of the SARS-CoV-2 proteins have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate viral replication, overall virulence, and viral genomic RNA reproduction. We sought to better characterize the SARS-CoV-2 Nsp9 and subsequently solved its X-ray crystal structure, in an apo form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The SARS-CoV-2 Nsp9 structure revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted the relative juxtapositioning of the monomers within the homodimer. We have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure, and identified a peptide-binding site that warrants further study to understanding Nsp9 function.
RESUMO
T cell receptors (TCRs) recognize antigens presented by major histocompatibility complex (MHC) and MHC class I-like molecules. We describe a diverse population of human γδ T cells isolated from peripheral blood and tissues that exhibit autoreactivity to the monomorphic MHC-related protein 1 (MR1). The crystal structure of a γδTCR-MR1-antigen complex starkly contrasts with all other TCR-MHC and TCR-MHC-I-like complex structures. Namely, the γδTCR binds underneath the MR1 antigen-binding cleft, where contacts are dominated by the MR1 α3 domain. A similar pattern of reactivity was observed for diverse MR1-restricted γδTCRs from multiple individuals. Accordingly, we simultaneously report MR1 as a ligand for human γδ T cells and redefine the parameters for TCR recognition.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Cristalografia por Raios X , Células HEK293 , Antígenos de Histocompatibilidade Classe I/química , Humanos , Antígenos de Histocompatibilidade Menor/química , Domínios Proteicos , Receptores de Antígenos de Linfócitos T gama-delta/químicaRESUMO
The interaction between natural killer (NK) cell inhibitory receptors and their cognate ligands constitutes a key mechanism by which healthy tissues are protected from NK cell-mediated lysis. However, self-ligand recognition remains poorly understood within the prototypical NKR-P1 receptor family. Here we report the structure of the inhibitory NKR-P1B receptor bound to its cognate host ligand, Clr-b. NKR-P1B and Clr-b interact via a head-to-head docking mode through an interface that includes a large array of polar interactions. NKR-P1B:Clr-b recognition is extremely sensitive to mutations at the heterodimeric interface, with most mutations severely impacting both Clr-b binding and NKR-P1B receptor function to implicate a low affinity interaction. Within the structure, two NKR-P1B:Clr-b complexes are cross-linked by a non-classic NKR-P1B homodimer, and the disruption of homodimer formation abrogates Clr-b recognition. These data provide an insight into a fundamental missing-self recognition system and suggest an avidity-based mechanism underpins NKR-P1B receptor function.
Assuntos
Lectinas Tipo C/química , Subfamília B de Receptores Semelhantes a Lectina de Células NK/química , Receptores Imunológicos/química , Receptores de Células Matadoras Naturais/química , Animais , Proteínas de Transporte , Cristalografia por Raios X , Células HEK293 , Humanos , Lectinas Tipo C/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Receptores Imunológicos/genética , Receptores de Células Matadoras Naturais/genética , Difração de Raios XRESUMO
Human leukocyte antigen (HLA)-I molecules generally bind short peptides (8-10 amino acids), although extended HLA-I restricted peptides (>10 amino acids) can be presented to T cells. However, the function of such extended HLA-I epitopes in tumour immunity, and how they would be recognised by T-cell receptors (TCR) remains unclear. Here we show that the structures of two distinct TCRs (TRAV4+TRAJ21+-TRBV28+TRBJ2-3+ and TRAV4 + TRAJ8+-TRBV9+TRBJ2-1+), originating from a polyclonal T-cell repertoire, bind to HLA-B*07:02, presenting a 13-amino-acid-long tumour-associated peptide, NY-ESO-160-72. Comparison of the structures reveals that the two TCRs differentially binds NY-ESO-160-72-HLA-B*07:02 complex, and induces differing extent of conformational change of the NY-ESO-160-72 epitope. Accordingly, polyclonal TCR usage towards an extended HLA-I restricted tumour epitope translates to differing TCR recognition modes, whereby extensive flexibility at the TCR-pHLA-I interface engenders recognition.
Assuntos
Antígeno HLA-B7/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Antígeno HLA-B7/química , Antígeno HLA-B7/genética , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/genética , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
In the structural analysis of proteins via X-ray diffraction, a rate-limiting step is in favourable nucleation, a problematic obstacle in successful generation of protein crystals. Here graphene and graphene oxide were applied to protein crystallisation trials, offering improvements in crystalline output and nucleation.