ABSTRACT
Insights into mechanisms driving either activation or inhibition of immune response are crucial in understanding the pathology of various diseases. The differentiation of viral from endogenous RNA in the cytoplasm by pattern-recognition receptors, such as retinoic acid-inducible gene I (RIG-I), is one of the essential paths for timely activation of an antiviral immune response through induction of type I interferons (IFN). In this mini-review, we describe the most recent developments centered around RIG-I's structure and mechanism of action. We summarize the paradigm-changing work over the past few years that helped us better understand RIG-I's monomeric and oligomerization states and their role in conveying immune response. We also discuss potential applications of the modulation of the RIG-I pathway in preventing autoimmune diseases or induction of immunity against viral infections. Overall, our review aims to summarize innovative research published in the past few years to help clarify questions that have long persisted around RIG-I.
Subject(s)
DEAD Box Protein 58 , Receptors, Immunologic , Humans , DEAD Box Protein 58/metabolism , DEAD Box Protein 58/immunology , DEAD Box Protein 58/genetics , DEAD Box Protein 58/chemistry , Receptors, Immunologic/chemistry , Receptors, Immunologic/metabolism , Animals , Virus Diseases/immunology , Interferon Type I/immunology , Interferon Type I/metabolism , Signal Transduction , Protein Multimerization , Immunity, InnateABSTRACT
Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor that can be targeted for inducing potent adjuvant effects. Mincle can recognize trehalose dimycolate and related glycolipids. Here, we present a protocol to identify the ligand binding mode of Mincle. We describe steps for preparing labeled Mincle ectodomain, data acquisition, and analysis of nuclear magnetic resonance experiments using non-detergent sulfobetaine-195. This protocol can be applied to other protein-ligand interactions that have aggregation problems for complex formation. For complete details on the use and execution of this protocol, please refer to Furukawa et al.1.
Subject(s)
Lectins, C-Type , Lectins, C-Type/metabolism , Lectins, C-Type/chemistry , Ligands , Binding Sites , Humans , Magnetic Resonance Spectroscopy/methods , Protein Binding , Receptors, Immunologic/chemistry , Receptors, Immunologic/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular/methodsABSTRACT
Nectin and nectin-like (Necl) co-receptor axis, comprised of receptors DNAM-1, TIGIT, CD96, PVRIG, and nectin/Necl ligands, is gaining prominence in immuno-oncology. Within this axis, the inhibitory receptor PVRIG recognizes Nectin-2 with high affinity, but the underlying molecular basis remains unknown. By determining the crystal structure of PVRIG in complex with Nectin-2, we identified a unique CC' loop in PVRIG, which complements the double-lock-and-key binding mode and contributes to its high affinity for Nectin-2. The association of the corresponding charged residues in the F-strands explains the ligand selectivity of PVRIG toward Nectin-2 but not for Necl-5. Moreover, comprehensive comparisons of the binding capacities between co-receptors and ligands provide innovative insights into the intra-axis immunoregulatory mechanism. Taken together, these findings broaden our understanding of immune recognition and regulation mediated by nectin/Necl co-receptors and provide a rationale for the development of immunotherapeutic strategies targeting the nectin/Necl axis.
Subject(s)
Models, Molecular , Nectins , Protein Binding , Receptors, Cell Surface , Humans , Binding Sites , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/immunology , Crystallography, X-Ray , Ligands , Nectins/metabolism , Nectins/chemistry , Receptors, Immunologic/metabolism , Receptors, Immunologic/chemistry , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolismABSTRACT
TIGIT is mainly expressed on T cells and is an inhibitory checkpoint receptor that binds to its ligand PVR in the tumor microenvironment. Anti-TIGIT monoclonal antibodies (mAbs) such as Ociperlimab and Tiragolumab block the TIGIT-PVR interaction and are in clinical development. However, the molecular blockade mechanism of these mAbs remains elusive. Here, we report the crystal structures of TIGIT in complex with Ociperlimab_Fab and Tiragolumab_Fab revealing that both mAbs bind TIGIT with a large steric clash with PVR. Furthermore, several critical epitopic residues are identified. Interestingly, the binding affinity of Ociperlimab toward TIGIT increases approximately 17-fold when lowering the pH from 7.4 to 6.0. Our structure shows a strong electrostatic interaction between ASP103HCDR3 and HIS76TIGIT explaining the pH-responsive mechanism of Ociperlimab. In contrast, Tiragolumab does not show an acidic pH-dependent binding enhancement. Our results provide valuable information that could help to improve the efficacy of therapeutic antibodies for cancer treatment.
Subject(s)
Models, Molecular , Protein Binding , Receptors, Immunologic , Hydrogen-Ion Concentration , Humans , Receptors, Immunologic/metabolism , Receptors, Immunologic/chemistry , Crystallography, X-Ray , Antibodies, Monoclonal/chemistry , Binding Sites , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/immunologyABSTRACT
Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.
Subject(s)
Adenosine Triphosphate , Arabidopsis , NAD , Nicotiana , Phase Separation , Plant Proteins , Protein Domains , Adenosine Triphosphate/metabolism , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/immunology , Arabidopsis Proteins/metabolism , Cell Death , Mutation , NAD/metabolism , Nicotiana/genetics , Nicotiana/immunology , Nicotiana/metabolism , NLR Proteins/chemistry , NLR Proteins/genetics , NLR Proteins/immunology , NLR Proteins/metabolism , Plant Diseases/immunology , Plant Immunity/genetics , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/immunology , Plant Proteins/metabolism , Promoter Regions, Genetic , Protein Domains/genetics , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism , Signal Transduction , Toll-Like Receptors/chemistry , Receptors, Interleukin-1/chemistryABSTRACT
Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive "dimer of heterodimers" and that structural heterogeneity is expected even among alleles of closely related paired NLRs.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Chitin Synthase , NLR Proteins , Plant Diseases , Plant Immunity , Receptors, Immunologic , Alleles , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chitin Synthase/chemistry , Chitin Synthase/genetics , Chitin Synthase/metabolism , Mutation , NLR Proteins/chemistry , NLR Proteins/genetics , NLR Proteins/metabolism , Plant Diseases/genetics , Plant Diseases/immunology , Plant Immunity/genetics , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Protein MultimerizationABSTRACT
With the expansion of ICP-MS application into the field of bioanalysis, there is an urgent need for novel element tags today. Here, we report the design of a dual-element Ir-Eu tag, opening the door to simultaneous fluorescent imaging and ICP-MS quantification. The ratio of 153Eu/193Ir may serve as a precision control of the labeling process, allowing internal validation of the quantitative results obtained. As for SIRPα and its host cell analysis exemplified here, the Ir-Eu tag demonstrated superior figures of ICP-MS quantification with the LOD (3σ) down to 0.5 (153Eu) and 1.1 (193Ir) pM SIRPα and 220 (153Eu) and 830 (193Ir) RAW264.7 cells more than 130 times more sensitive compared with the LOD (3σ) of 65.2 pM SIRPα at 612 nm using fluorometry. Not limited to these demonstrations, we believe that the design ideas of the dual Ir-Eu tags should be applicable to various cases of bioanalysis when dual optical profiling and ICP-MS quantification are indispensable.
Subject(s)
Mass Spectrometry , Fluorometry , Mass Spectrometry/methods , Spectrum Analysis , Iridium/chemistry , Europium/chemistry , Fluorescent Dyes/chemistry , Animals , Mice , Receptors, Immunologic/analysis , Receptors, Immunologic/chemistry , RAW 264.7 CellsABSTRACT
CD300A is a member of the CD300 immunoglobulin (Ig)-like receptor family consisting of eight molecules in humans, all of which contain one Ig-like domain in the extracellular portion. Upon binding its ligand phosphatidylserine or phosphatidylethanolamine, CD300A mediates an inhibitory signal through the immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic portion. The CD300 family molecules are highly homologous to each other. In addition, CD300A has a single nucleotide polymorphism (rs2272111), which is a nonsense mutation encoding glutamine (CD300AQ111) instead of arginine (CD300AR111) at residue 111 in the Ig-like domain of CD300A. In this study, we successfully generated monoclonal antibodies (mAbs) specific to either CD300AR111 or CD300AQ111 or both. These mAbs are useful for the analysis of CD300A genotype by flow cytometry and the development of an antibody drug for the treatment of various diseases.
Subject(s)
Antibodies, Monoclonal , Receptors, Immunologic , Humans , Receptors, Immunologic/genetics , Receptors, Immunologic/chemistry , Antigens, CD/geneticsABSTRACT
RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.
Subject(s)
DEAD Box Protein 58 , Isoleucine , Receptors, Immunologic , DEAD Box Protein 58/chemistry , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , Immune Tolerance , Isoleucine/genetics , RNA, Double-Stranded/genetics , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Humans , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolismABSTRACT
IMPORTANCE: A common hypothesis holds that bats (order Chiroptera) are outstanding reservoirs for zoonotic viruses because of a special antiviral interferon (IFN) system. However, functional studies about key components of the bat IFN system are rare. RIG-I is a cellular sensor for viral RNA signatures that activates the antiviral signaling chain to induce IFN. We cloned and functionally characterized RIG-I genes from two species of the suborders Yangochiroptera and Yinpterochiroptera. The bat RIG-Is were conserved in their sequence and domain organization, and similar to human RIG-I in (i) mediating virus- and IFN-activated gene expression, (ii) antiviral signaling, (iii) temperature dependence, and (iv) recognition of RNA ligands. Moreover, RIG-I of Rousettus aegyptiacus (suborder Yinpterochiroptera) and of humans were found to recognize SARS-CoV-2 infection. Thus, members of both bat suborders encode RIG-Is that are comparable to their human counterpart. The ability of bats to harbor zoonotic viruses therefore seems due to other features.
Subject(s)
Chiroptera , Receptors, Retinoic Acid , SARS-CoV-2 , Animals , Humans , Chiroptera/metabolism , COVID-19 , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , SARS-CoV-2/physiology , Viruses , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolismABSTRACT
The intricate complex system of the differentiation 47 (CD47) and the signal-regulatory protein alpha (SIRPα) cluster is a crucial target for cancer immunotherapy. Although the conformational state of the CD47-SIRPα complex has been revealed through crystallographic studies, further characterization is needed to fully understand the binding mechanism and to identify the hot spot residues involved. In this study, molecular dynamics (MD) simulations were carried out for the complexes of CD47 with two SIRPα variants (SIRPαv1, SIRPαv2) and the commercially available anti-CD47 monoclonal antibody (B6H12.2). The calculated binding free energy of CD47-B6H12.2 is lower than that of CD47-SIRPαv1 and CD47-SIRPαv2 in all the three simulations, indicating that CD47-B6H12.2 has a higher binding affinity than the other two complexes. Moreover, the dynamical cross-correlation matrix reveals that the CD47 protein shows more correlated motions when it binds to B6H12.2. Significant effects were observed in the energy and structural analyses of the residues (Glu35, Tyr37, Leu101, Thr102, Arg103) in the C strand and FG region of CD47 when it binds to the SIRPα variants. The critical residues (Leu30, Val33, Gln52, Lys53, Thr67, Arg69, Arg95, and Lys96) were identified in SIRPαv1 and SIRPαv2, which surround the distinctive groove regions formed by the B2C, C'D, DE, and FG loops. Moreover, the crucial groove structures of the SIRPα variants shape into obvious druggable sites. The C'D loops on the binding interfaces undergo notable dynamical changes throughout the simulation. For B6H12.2, the residues Tyr32LC, His92LC, Arg96LC, Tyr32HC, Thr52HC, Ser53HC, Ala101HC, and Gly102HC in its initial half of the light and heavy chains exhibit obvious energetic and structural impacts upon binding with CD47. The elucidation of the binding mechanism of SIRPαv1, SIRPαv2, and B6H12.2 with CD47 could provide novel perspectives for the development of inhibitors targeting CD47-SIRPα.
Subject(s)
Molecular Dynamics Simulation , Neoplasms , Humans , Receptors, Immunologic/chemistry , Antigens, Differentiation/chemistry , CD47 Antigen/genetics , CD47 Antigen/chemistry , Antibodies, Monoclonal , Immunotherapy , Phagocytosis , Neoplasms/metabolismABSTRACT
Natural killer (NK) cells play an important role in the innate immune response against tumors and various pathogens such as viruses and bacteria. Their function is controlled by a wide array of activating and inhibitory receptors, which are expressed on their cell surface. Among them is a dimeric NKG2A/CD94 inhibitory transmembrane (TM) receptor which specifically binds to the non-classical MHC I molecule HLA-E, which is often overexpressed on the surface of senescent and tumor cells. Using the Alphafold 2 artificial intelligence system, we constructed the missing segments of the NKG2A/CD94 receptor and generated its complete 3D structure comprising extracellular (EC), TM, and intracellular regions, which served as a starting point for the multi-microsecond all-atom molecular dynamics simulations of the receptor with and without the bound HLA-E ligand and its nonameric peptide. The simulated models revealed that an intricate interplay of events is taking place between the EC and TM regions ultimately affecting the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) regions that host the point at which the signal is transmitted further down the inhibitory signaling cascade. Signal transduction through the lipid bilayer was also coupled with the changes in the relative orientation of the NKG2A/CD94 TM helices in response to linker reorganization, mediated by fine-tuned interactions in the EC region of the receptor, taking place after HLA-E binding. This research provides atomistic details of the cells' protection mechanism against NK cells and broadens the knowledge regarding the TM signaling of ITIM-bearing receptors.
Subject(s)
NK Cell Lectin-Like Receptor Subfamily C , Receptors, Immunologic , NK Cell Lectin-Like Receptor Subfamily C/metabolism , Receptors, Natural Killer Cell/metabolism , Receptors, Immunologic/chemistry , Receptors, Immunologic/metabolism , Ligands , Artificial Intelligence , Histocompatibility Antigens Class I/metabolism , Signal Transduction , Carrier Proteins/metabolism , HLA-E AntigensABSTRACT
AIM: Repetitive transcranial magnetic stimulation (rTMS) is one of the most effective and minimally invasive treatments for treatment-resistant depression (TRD). However, the mechanism underlying the therapeutic effects of rTMS in patients with TRD remains unclear. In recent years, the pathogenesis of depression has been closely associated with chronic inflammation and microglia are believed to play an important role in chronic inflammation. Triggering receptor expressed on myeloid cells-2 (TREM2) plays an important role in microglial neuroinflammatory regulation. In this study, we investigated the changes in peripheral soluble TREM2 (sTREM2) before and after rTMS treatment in patients with TRD. METHODS: Twenty-six patients with TRD were enrolled in this frequency (10 Hz) rTMS study. Depressive symptoms, cognitive function, and serum sTREM2 concentrations were measured at baseline and the end of the 6-week rTMS treatment. RESULTS: This study showed that rTMS ameliorated depressive symptoms and partially improved cognitive dysfunction in TRD. However, rTMS treatment did not alter serum sTREM2 levels. CONCLUSIONS: This is the first sTREM2 study in patients with TRD who underwent rTMS treatment. These results suggest that serum sTREM2 may not be relevant for the mechanism underlying the therapeutic effect of rTMS in patients with TRD. Future studies should confirm the present findings using a larger patient sample and a sham rTMS procedure, as well as CSF sTREM2. Furthermore, a longitudinal study should be conducted to clarify the effects of rTMS on sTREM2 levels.
Subject(s)
Depressive Disorder, Treatment-Resistant , Receptors, Immunologic , Transcranial Magnetic Stimulation , Female , Humans , Male , Middle Aged , Body Mass Index , Cognition , Depression/psychology , Depression/therapy , Depressive Disorder, Treatment-Resistant/psychology , Depressive Disorder, Treatment-Resistant/therapy , Longitudinal Studies , Receptors, Immunologic/blood , Receptors, Immunologic/chemistry , SmokingABSTRACT
Mechanical cues dynamically regulate membrane receptors functions to trigger various physiological and pathological processes from viral invasion to immune defense. These cues mainly include various types of dynamic mechanical forces and the spatial confinement of plasma membrane. However, the molecular mechanisms of how they couple with biochemical cues in regulating membrane receptors functions still remain mysterious. Here, we review recent advances in methodologies of single-molecule biomechanical techniques and in novel biomechanical regulatory mechanisms of critical ligand recognition of viral and immune receptors including SARS-CoV-2 spike protein, T cell receptor (TCR) and other co-stimulatory immune receptors. Furthermore, we provide our perspectives of the general principle of how force-dependent kinetics determine the dynamic functions of membrane receptors and of biomechanical-mechanism-driven SARS-CoV-2 neutralizing antibody design and TCR engineering for T-cell-based therapies.
Subject(s)
Receptors, Immunologic , Single Molecule Imaging , Virus Internalization , Humans , Receptors, Antigen, T-Cell/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Receptors, Immunologic/chemistry , Microscopy, Atomic Force , ImmunityABSTRACT
Background: Targeting the CD47/SIRPα signaling pathway represents a novel approach to enhance anti-tumor immunity. However, the crystal structure of the CD47/SIRPα has not been fully studied. This study aims to analyze the structure interface of the complex of CD47 and IMM01, a novel recombinant SIRPα-Fc fusion protein. Methods: IMM01-Fab/CD47 complex was crystalized, and diffraction images were collected. The complex structure was determined by molecular replacement using the program PHASER with the CD47-SIRPαv2 structure (PDB code 2JJT) as a search model. The model was manually built using the COOT program and refined using TLS parameters in REFMAC from the CCP4 program suite. Results: Crystallization and structure determination analysis of the interface of IMM01/CD47 structure demonstrated CD47 surface buried by IMM01. Comparison with the literature structure (PDB ID 2JJT) showed that the interactions of IMM01/CD47 structure are the same. All the hydrogen bonds that appear in the literature structure are also present in the IMM01/CD47 structure. These common hydrogen bonds are stable under different crystal packing styles, suggesting that these hydrogen bonds are important for protein binding. In the structure of human CD47 in complex with human SIRPα, except SER66, the amino acids that form hydrogen bonds are all conserved. Furthermore, comparing with the structure of PDB ID 2JJT, the salt bridge interaction from IMM01/CD47 structure are very similar, except the salt bridge bond between LYS53 in IMM01 and GLU106 in CD47, which only occurs between the B and D chains. However, as the side chain conformation of LYS53 in chain A is slightly different, the salt bridge bond is absent between the A and C chains. At this site between chain A and chain C, there are a salt bridge bond between LYS53 (A) and GLU104 (C) and a salt bridge bond between HIS56 (A) and GLU106 (C) instead. According to the sequence alignment results of SIRPα, SIRPß and SIRPγ in the literature of PDB ID 2JJT, except ASP100, the amino acids that form common salt bridge bonds are all conserved. Conclusion: Our data demonstrated crystal structure of the IMM01/CD47 complex and provides a structural basis for the structural binding interface and future clinical applications.
Subject(s)
Amino Acids , Antigens, Differentiation , CD47 Antigen , Receptors, Immunologic , Amino Acids/chemistry , Antigens, Differentiation/chemistry , CD47 Antigen/chemistry , Humans , Phagocytosis , Protein Binding , Receptors, Immunologic/chemistry , Recombinant Fusion Proteins/chemistryABSTRACT
The engagement of the herpesvirus entry mediator (HVEM, TNFRSF14) by the B and T lymphocyte attenuator (BTLA) represents a unique interaction between an activating receptor of the TNFR-superfamily and an inhibitory receptor of the Ig-superfamily. BTLA and HVEM have both been implicated in the regulation of human T cell responses, but their role is complex and incompletely understood. Here, we have used T cell reporter systems to dissect the complex interplay of HVEM with BTLA and its additional ligands LIGHT and CD160. Co-expression with LIGHT or CD160, but not with BTLA, induced strong constitutive signaling via HVEM. In line with earlier reports, we observed that in cis interaction of BTLA and HVEM prevented HVEM co-stimulation by ligands on surrounding cells. Intriguingly, our data indicate that BTLA mediated inhibition is not impaired in this heterodimeric complex, suggesting a dominant role of BTLA co-inhibition. Stimulation of primary human T cells in presence of HVEM ligands indicated a weak costimulatory capacity of HVEM potentially owed to its in cis engagement by BTLA. Furthermore, experiments with T cell reporter cells and primary T cells demonstrate that HVEM antibodies can augment T cell responses by concomitantly acting as checkpoint inhibitors and co-stimulation agonists.
Subject(s)
Receptors, Immunologic , Receptors, Tumor Necrosis Factor, Member 14 , T-Lymphocytes , Antigens, CD , B-Lymphocytes/metabolism , GPI-Linked Proteins , Humans , Ligands , Receptors, Immunologic/chemistry , Receptors, Immunologic/metabolism , Receptors, Tumor Necrosis Factor, Member 14/chemistry , Receptors, Tumor Necrosis Factor, Member 14/metabolism , Signal TransductionABSTRACT
Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.
Subject(s)
Calcium Channels , Cryoelectron Microscopy , NLR Proteins , Plant Proteins , Receptors, Immunologic , Triticum , Arabidopsis/immunology , Arabidopsis/metabolism , Arginine , Calcium Channels/chemistry , Calcium Channels/immunology , Calcium Channels/metabolism , Cations/metabolism , Leucine , NLR Proteins/chemistry , NLR Proteins/immunology , NLR Proteins/metabolism , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Immunity , Plant Proteins/chemistry , Plant Proteins/immunology , Plant Proteins/metabolism , Receptors, Immunologic/chemistry , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism , Triticum/immunology , Triticum/metabolism , Amino Acid Motifs , Conserved Sequence , ElectrophysiologyABSTRACT
Human roundabout 1 (hRobo1) is an extracellular receptor glycoprotein that plays important roles in angiogenesis, organ development, and tumor progression. Interaction between hRobo1 and heparan sulfate (HS) has been shown to be essential for its biological activity. To better understand the effect of HS binding we engineered a lanthanide-binding peptide sequence (Loop) into the Ig2 domain of hRobo1. Native mass spectrometry was used to verify that loop introduction did not inhibit HS binding or conformational changes previously suggested by gas phase ion mobility measurements. NMR experiments measuring long-range pseudocontact shifts were then performed on 13C-methyl labeled hRobo1-Ig1-2-Loop in HS-bound and unbound forms. The magnitude of most PCSs for methyl groups in the Ig1 domain increase in the bound state confirming a change in the distribution of interdomain geometries. A grid search over Ig1 orientations to optimize the fit of data to a single conformer for both forms produced two similar structures, both of which differ from existing X-ray crystal structures and structures inferred from gas-phase ion mobility measurements. The structures and degree of fit suggest that the hRobo1-Ig1-2 structure changes slightly and becomes more rigid on HS binding. This may have implications for Robo-Slit signaling.
Subject(s)
Nerve Tissue Proteins/chemistry , Receptors, Immunologic/chemistry , Heparitin Sulfate/metabolism , Humans , Magnetic Resonance Spectroscopy , Nerve Tissue Proteins/metabolism , Receptors, Immunologic/metabolism , Signal Transduction , Roundabout ProteinsABSTRACT
Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL-Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions.
Subject(s)
Calcium-Binding Proteins , Nerve Tissue Proteins , Receptors, Immunologic , Binding Sites , Calcium-Binding Proteins/chemistry , Ligands , Nerve Tissue Proteins/chemistry , Protein Domains , Receptors, Immunologic/chemistryABSTRACT
The association between two intrinsically disordered proteins (IDPs) may produce a fuzzy complex characterized by a high binding affinity, similar to that found in the ultrastable complexes formed between two well-structured proteins. Here, using coarse-grained simulations, we quantified the biophysical forces driving the formation of such fuzzy complexes. We found that the high-affinity complex formed between the highly and oppositely charged H1 and ProTα proteins is sensitive to electrostatic interactions. We investigated 52 variants of the complex by swapping charges between the two oppositely charged proteins to produce sequences whose negatively or positively charged residue content was more homogeneous or heterogenous (i.e., polyelectrolytic or polyampholytic, having higher or lower absolute net charges, respectively) than the wild type. We also changed the distributions of oppositely charged residues within each participating sequence to produce variants in which the charges were segregated or well mixed. Both types of changes significantly affect binding affinity in fuzzy complexes, which is governed by both enthalpy and entropy. The formation of H1-ProTa is supported by an increase in configurational entropy and by entropy due to counterion release. The latter can be twice as large as the former, illustrating the dominance of counterion entropy in modulating the binding thermodynamics. Complexes formed between proteins with greater absolute net charges are more stable, both enthalpically and entropically, indicating that enthalpy and entropy have a mutually reinforcing effect. The sensitivity of the thermodynamics of the complex to net charge and the charge pattern within each of the binding constituents may provide a means to achieve binding specificity between IDPs.