RESUMEN
Robust and precise tools are needed to enhance the functionality and resilience of synthetic nanoarchitectures. Here, we have employed directed evolution and rational design to build a fast-acting molecular superglue from a bacterial adhesion protein. We have generated the SnoopLigase2 coupling system, a genetically encoded route for efficient transamidation between SnoopTag2 and DogTag2 peptides. Each peptide was selected for rapid reaction by phage display screening. The optimized set allows more than 99% completion and is compatible with diverse buffers, pH values, and temperatures, accelerating the reaction over 1000-fold. SnoopLigase2 directs a specific reaction in the mammalian secretory pathway, allowing covalent display on the plasma membrane. Transglutaminase 2 (TG2) has a network of interactions and substrates amidst the mammalian cell surface and extracellular matrix. We expressed a modified TG2 with resistance to oxidative inactivation and minimal self-reactivity. SnoopLigase2 enables TG2 functionalization with transforming growth factor alpha (TGFα) in routes that would be impossible through genetic fusion. The TG2:TGFα conjugate retained transamidase activity, stably anchored TGFα for signal activation in the extracellular environment, and reprogrammed cell behavior. This modular toolbox should create new opportunities for molecular assembly, both for novel biomaterials and complex cellular environments.
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Factor de Crecimiento Transformador alfa , Transglutaminasas , Animales , Transglutaminasas/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Péptidos/química , Membrana Celular/metabolismo , Mamíferos/metabolismoRESUMEN
Much of life's complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit. Prototypes of infinite affinity pairs have been achieved using nonnatural reactive groups. After library-based evolution and rational design, here we establish a peptide-protein pair composed of the regular 20 amino acids that link together through an amide bond at a rate approaching the diffusion limit. Reaction occurs in a few minutes with both partners at low nanomolar concentration. Stopped flow fluorimetry illuminated the conformational dynamics involved in docking and reaction. Hydrogen-deuterium exchange mass spectrometry gave insight into the conformational flexibility of this split protein and the process of enhancing its reaction rate. We applied this reactive pair for specific labeling of a plasma membrane target in 1 min on live mammalian cells. Sensitive and specific detection was also confirmed by Western blot in a range of model organisms. The peptide-protein pair allowed reconstitution of a critical mechanotransmitter in the cytosol of mammalian cells, restoring cell adhesion and migration. This simple genetic encoding for rapid irreversible reaction should provide diverse opportunities to enhance protein function by rapid detection, stable anchoring, and multiplexing of protein functionality.
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Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE.
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Antiasmáticos/farmacología , Inmunoglobulina E/metabolismo , Modelos Moleculares , Omalizumab/farmacología , Receptores de IgE/antagonistas & inhibidores , Sitio Alostérico , Sustitución de Aminoácidos , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoglobulina E/química , Inmunoglobulina E/genética , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/farmacología , Omalizumab/química , Omalizumab/genética , Omalizumab/metabolismo , Docilidad , Mutación Puntual , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Replegamiento Proteico , Receptores de IgE/química , Receptores de IgE/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Solubilidad , Resonancia por Plasmón de SuperficieRESUMEN
The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 µM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 µM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.
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Antígenos CD/inmunología , Antígenos/inmunología , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Animales , Antígenos/química , Antígenos/genética , Antígenos CD/química , Antígenos CD/genética , Sitios de Unión , Butirofilinas , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Cristalografía por Rayos X , Difosfatos/farmacología , Difosfonatos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica/inmunología , Hemiterpenos/farmacología , Humanos , Imidazoles/farmacología , Activación de Linfocitos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Modelos Moleculares , Compuestos Organofosforados/farmacología , Fosfoproteínas/química , Fosfoproteínas/genética , Plaquinas/química , Plaquinas/genética , Plaquinas/inmunología , Cultivo Primario de Células , Unión Proteica , Receptores de Antígenos de Linfocitos T gamma-delta/química , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Transducción de Señal , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Técnicas del Sistema de Dos Híbridos , Ácido ZoledrónicoRESUMEN
SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram-positive bacterial adhesin. In this work, we established a phage-display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.
RESUMEN
Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcεRI and CD23 (FcεRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcεRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca(2+)-free and Ca(2+)-bound forms, as well as the crystal structure of the Ca(2+)-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). Together with site-directed mutagenesis, the crystal structures of four Ca(2+) ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca(2+) binds at the principal and evolutionarily conserved binding site in CD23. Ca(2+) binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca(2+)-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca(2+) brings an extra degree of modulation to CD23 function.
Asunto(s)
Linfocitos B/metabolismo , Calcio/metabolismo , Inmunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Unión Competitiva , Calorimetría , Cristalografía por Rayos X , Ciclofilina A/metabolismo , Entropía , Humanos , Inmunoglobulina E/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de IgE/química , Receptores de IgE/genética , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
Bispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry.
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Anticuerpos Biespecíficos , Epítopos , Péptido HidrolasasRESUMEN
Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Neutralizantes/inmunología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/química , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/virología , Humanos , Vacunación/métodos , Ratones , Nanopartículas/química , FemeninoRESUMEN
IgE binding to its high affinity receptor FcεRI on mast cells and basophils is a key step in the mechanism of allergic disease and a target for therapeutic intervention. Early indications that IgE adopts a bent structure in solution have been confirmed by recent x-ray crystallographic studies of IgEFc, which further showed that the bend, contrary to expectation, is enhanced in the crystal structure of the complex with receptor. To investigate the structure of IgEFc and its conformational changes that accompany receptor binding in solution, we created a Förster resonance energy transfer (FRET) biosensor using biologically encoded fluorescent proteins fused to the N- and C-terminal IgEFc domains (Cε2 and Cε4, respectively) together with the theoretical basis for quantitating its behavior. This revealed not only that the IgEFc exists in a bent conformation in solution but also that the bend is indeed enhanced upon FcεRI binding. No change in the degree of bending was seen upon binding to the B cell receptor for IgE, CD23 (FcεRII), but in contrast, binding of the anti-IgE therapeutic antibody omalizumab decreases the extent of the bend, implying a conformational change that opposes FcεRI engagement. HomoFRET measurements further revealed that the (Cε2)(2) and (Cε4)(2) domain pairs behave as rigid units flanking the conformational change in the Cε3 domains. Finally, modeling of the accessible conformations of the two Fab arms in FcεRI-bound IgE revealed a mutual exclusion not seen in IgG and Fab orientations relative to the membrane that may predispose receptor-bound IgE to cross-linking by allergens.
Asunto(s)
Alérgenos/análisis , Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Inmunoglobulina E/química , Fragmentos Fc de Inmunoglobulinas/química , Receptores de IgE/química , Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales Humanizados/química , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Inmunoglobulina E/genética , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Omalizumab , Receptores de IgE/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection.
RESUMEN
There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.
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Proteínas Luminiscentes/química , Oxidorreductasas/química , Péptidos/química , Células Cultivadas , Escherichia coli/citología , Humanos , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Oxidorreductasas/metabolismo , Péptidos/metabolismo , Conformación Proteica , SolubilidadRESUMEN
Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin.MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition.
Asunto(s)
Complemento C1q/genética , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Ingeniería de Proteínas , Proteína A Asociada a Surfactante Pulmonar/genética , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Secuencia de Aminoácidos , Animales , Activación de Complemento , Complemento C1q/metabolismo , Lectina de Unión a Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Datos de Secuencia Molecular , Conformación Proteica , Proteína A Asociada a Surfactante Pulmonar/química , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
The classical and lectin pathways of complement activation neutralize pathogens and stimulate key immunological processes. Both pathways are initiated by collagen-containing, soluble pattern recognition molecules associated with specific serine proteases. In the classical pathway, C1q binds to Ab-Ag complexes or bacterial surfaces to activate C1r and C1s. In the lectin pathway, mannan-binding lectin and ficolins bind to carbohydrates on pathogens to activate mannan-binding lectin-associated serine protease 2. To characterize the interactions leading to classical pathway activation, we have analyzed binding between human C1q, C1r, and C1s, which associate to form C1, using full-length and truncated protease components. We show that C1r and C1s bind to C1q independently. The CUB1-epidermal growth factor fragments contribute most toward binding, but CUB2 of C1r, but not of C1s, is also important. Each C1rs tetramer presents a total of six binding sites, one for each of the collagenous domains of C1q. We also demonstrate that subcomponents of the lectin and classical pathways cross-interact. Thus, although the stoichiometries of complexes differ, interactions are analogous, with equivalent contacts between recognition and protease subcomponents. Importantly, these new data are contrary to existing models of C1 and enable us to propose a new model using mannan-binding lectin-mannan-binding lectin-associated serine protease interactions as a template.
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Proteínas del Sistema Complemento/metabolismo , Lectinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/genética , Humanos , Cinética , Lectinas/química , Lectinas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
The newly identified tripartite motif (TRIM) family of proteins mediate innate immunity and other critical cellular functions. Here we show that TRIM21, which mediates the autoimmune diseases rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome, is a previously undescribed IgG receptor with a binding mechanism unlike known mammalian Fcgamma receptors. TRIM21 simultaneously targets conserved hot-spot residues on both Ig domains of the Fc fragment using a PRYSPRY domain with a preformed multisite interface. The binding sites on both TRIM21 and Fc are highly conserved to the extent that the proteins are functionally interchangeable through murine, canine, primate, and human species. Pre-steady-state analysis exposes mechanistic conservation at the level of individual residues, which make the same energetic and kinetic contributions to binding despite varying in sequence. Together, our results reveal that TRIM21 is a previously undescribed type of IgG receptor based on a non-Ig scaffold whose interaction at the fundamental level-structural, thermodynamic, and kinetic-is evolutionarily conserved.
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Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Receptores de IgG/química , Ribonucleoproteínas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Humanos , Inmunoglobulina G/inmunología , Cinética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Estructura Terciaria de Proteína , Receptores de IgG/genética , Receptores de IgG/inmunología , Ribonucleoproteínas/genética , Ribonucleoproteínas/inmunología , Alineación de Secuencia , TermodinámicaRESUMEN
There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.
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Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Péptidos/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , Línea Celular , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , PorcinosRESUMEN
Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein analysis or application. Here we review SpyTag technology, now used in hundreds of publications or patents, and its potential for detecting and controlling protein behaviour. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded. New variants of this pair allow reaction at a rate approaching the diffusion limit, while reversible versions allow purification of SpyTagged proteins or tuned dynamic interaction inside cells. Anchoring of SpyTag-linked proteins has been established to diverse nanoparticles or surfaces, including gold, graphene and the air/water interface. SpyTag/SpyCatcher is mechanically stable, so is widely used for investigating protein folding and force sensitivity. A toolbox of scaffolds allows SpyTag-fusions to be assembled into defined multimers, from dimers to 180-mers, or unlimited 1D, 2D or 3D networks. Icosahedral multimers are being evaluated for vaccination against malaria, HIV and cancer. For enzymes, Spy technology has increased resilience, promoted substrate channelling, and assembled hydrogels for continuous flow biocatalysis. Combinatorial increase in functionality has been achieved through modular derivatisation of antibodies, light-emitting diodes or viral vectors. In living cells, SpyTag allowed imaging of protein trafficking, retargeting of CAR-T cell killing, investigation of heart contraction, and control of nucleosome position. The simple genetic encoding and rapid irreversible reaction provide diverse opportunities to enhance protein function. We describe limitations as well as future directions.
RESUMEN
New biological properties can stem from the freedom to link, multimerize, or multiplex protein building blocks. The peptide SpyTag on one protein irreversibly reacts with SpyCatcher on another protein, through spontaneous isopeptide bond formation. Reaction is specific in a wide range of cellular environments and all components are genetically encoded, making this chemistry accessible to molecular biologists. SpyTag/SpyCatcher has been widely used for enzyme immobilization, colocalization of different enzymatic activities, and increasing enzyme resilience. Here we present routes and advice for efficient design, expression, and purification of SpyTag/SpyCatcher constructs in bacterial and eukaryotic environments, including the latest 002 variants, and how to analyze reaction efficiency. The SpyInfo webpage collates the different publications and patents using SpyTag/SpyCatcher, while the SpyBank database lists their sequences and expression routes. The ability of SpyTag/SpyCatcher to react in a broad range of situations creates diverse opportunities for augmenting the function of enzymes and other biomolecules.
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Péptidos/genética , Western Blotting/métodos , Bases de Datos de Proteínas , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expresión Génica , Células HEK293 , Humanos , Péptidos/química , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Peptide tags are a key resource, introducing minimal change while enabling a consistent process to purify diverse proteins. However, peptide tags often provide minimal benefit post-purification. We previously designed SpyTag, forming an irreversible bond with its protein partner SpyCatcher. SpyTag provides an easy route to anchor, bridge or multimerize proteins. Here we establish Spy&Go, enabling protein purification using SpyTag. Through rational engineering we generated SpyDock, which captures SpyTag-fusions and allows efficient elution. Spy&Go enabled sensitive purification of SpyTag-fusions from Escherichia coli, giving superior purity than His-tag/nickel-nitrilotriacetic acid. Spy&Go allowed purification of mammalian-expressed, N-terminal, C-terminal or internal SpyTag. As an oligomerization toolbox, we established a panel of SpyCatcher-linked coiled coils, so SpyTag-fusions can be dimerized, trimerized, tetramerized, pentamerized, hexamerized or heptamerized. Assembling oligomers for Death Receptor 5 stimulation, we probed multivalency effects on cancer cell death. Spy&Go, combined with simple oligomerization, should have broad application for exploring multivalency in signaling.
Asunto(s)
Biotecnología/métodos , Proteínas/aislamiento & purificación , Proteínas de Escherichia coli , Modelos Moleculares , Péptidos/química , Proteínas PeriplasmáticasRESUMEN
The antibody IgE plays a central role in allergic disease mechanisms. Its effector functions are controlled through interactions between the Fc region and two principal cell surface receptors FcεRI and CD23. The interaction with FcεRI is primarily responsible for allergic sensitization and the inflammatory response, while IgE binding to CD23 is involved in the regulation of IgE synthesis and allergen transcytosis. Here we present the crystal structure of a CD23/IgE-Fc complex and conduct isothermal titration calorimetric binding studies. Two lectin-like "head" domains of CD23 bind to IgE-Fc with affinities that differ by more than an order of magnitude, but the crystal structure reveals only one head bound to one of the two identical heavy-chains in the asymmetrically bent IgE-Fc. These results highlight the subtle interplay between receptor binding sites in IgE-Fc and their affinities, the understanding of which may be exploited for therapeutic intervention in allergic disease.
Asunto(s)
Linfocitos B/inmunología , Sitios de Unión/inmunología , Inmunoglobulina E/inmunología , Receptores de IgE/inmunología , Alérgenos/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Unión Proteica/inmunología , Transcitosis/inmunologíaRESUMEN
The antibacterial activity of E colicin endonucleases (DNases) is counteracted by the binding of immunity proteins; the affinities of cognate and non-cognate complexes differing by up to ten orders of magnitude. Here, we address the mechanism of complex formation using a combination of protein engineering, pre-steady-state kinetics and isothermal titration calorimetry, in order to understand the underlying basis for specificity. Contrary to previous work, we show that a pre-equilibrium mechanism does not explain the binding kinetics. Instead, the data are best explained by a modified induced-fit mechanism where cognate and non-cognate complexes alike form a non-specific, conformationally dynamic encounter complex, most likely centred on conserved interactions at the interface. The dynamics appear to be an intrinsic property of the encounter complex where the proteins move relative to one another, thereby sampling different conformations rather than being "induced" by binding. This allows optimal alignment of interface specificity sites, without producing energetically costly conformational changes, essential for high-affinity binding. Importantly, specificity is achieved without slowing the rate of association, an important requirement for rapid inhibition of the colicin in the producing bacterial cell. A rigid-body rotation model is also consistent with the observation that specificity contacts in colicin-immunity protein complexes can involve different regions of the interface. Such a kinetic discrimination mechanism explains the ability of DNase-specific immunity proteins to display dual recognition specificity, wherein they are broadly cross-reactive yet are highly specific, achieving femtomolar binding affinities in complexes with their cognate DNases.