RESUMEN
Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7+ COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3ß loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Epítopos Inmunodominantes/inmunología , SARS-CoV-2/inmunología , Secuencia de Aminoácidos , Coronavirus/clasificación , Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/química , Reacciones Cruzadas , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígeno HLA-B7/química , Antígeno HLA-B7/genética , Antígeno HLA-B7/inmunología , Humanos , Epítopos Inmunodominantes/química , Memoria Inmunológica , Modelos Moleculares , Péptidos/química , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
Asunto(s)
Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Ubiquitina/metabolismo , Animales , Sitios de Unión , Chlorocebus aethiops , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/genética , Cristalografía por Rayos X , Citocinas/genética , Evaluación Preclínica de Medicamentos/métodos , Reposicionamiento de Medicamentos , Polarización de Fluorescencia , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Inhibidores de Proteasas/farmacología , Conformación Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Ubiquitinas/genética , Células VeroRESUMEN
A new high-pressure single-crystal diffraction setup has been designed and implemented at the Australian Synchrotron for collecting molecular and protein crystal structures. The setup incorporates a modified micro-Merrill-Bassett cell and holder designed specifically to fit onto the horizontal air-bearing goniometer, allowing high-pressure diffraction measurements to be collected with little to no modification of the beamline setup compared with ambient data collections. Compression data for the amino acid, L-threonine, and the protein, hen egg-white lysozyme, were collected, showcasing the capabilities of the setup.
Asunto(s)
Proteínas , Sincrotrones , Australia , Cristalografía por Rayos X , Proteínas/química , AminoácidosRESUMEN
MX2 is an in-vacuum undulator-based crystallography beamline at the 3â GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range 4.8-21â keV to a focal spot of 22 × 12â µm FWHM (H × V). At 13â keV the flux at the sample is 3.4 × 1012â photons s-1. The beamline endstation allows robotic handling of cryogenic samples via an updated SSRL SAM robot. This beamline is ideal for weakly diffracting hard-to-crystallize proteins, virus particles, protein assemblies and nucleic acids as well as smaller molecules such as inorganic catalysts and organic drug molecules. The beamline is now mature and has enjoyed a full user program for the last nine years. This paper describes the beamline status, plans for its future and some recent scientific highlights.
RESUMEN
MX1 is a bending-magnet crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range from 8 to 18 keV to a focal spot at the sample position of 120 µm FWHM. The beamline endstation and ancillary equipment facilitate local and remote access for both chemical and biological macromolecular crystallography. Here, the design of the beamline and endstation are discussed. The beamline has enjoyed a full user program for the last seven years and scientific highlights from the user program are also presented.
RESUMEN
As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of `microdomains'. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8â µm.
Asunto(s)
Ascaris suum/química , Proteínas de Unión a Ácidos Grasos/química , Animales , Cristalización , Cristalografía por Rayos X/instrumentación , Cristalografía por Rayos X/métodosRESUMEN
Interactions between a T cell receptor (TCR) and a peptide-major histocompatibility complex (pMHC) ligand are typically mediated by noncovalent bonds. By studying T cells expressing natural or engineered TCRs, here we describe covalent TCR-pMHC interactions that involve a cysteine-cysteine disulfide bond between the TCR and the peptide. By introducing cysteines into a known TCR-pMHC combination, we demonstrate that disulfide bond formation does not require structural rearrangement of the TCR or the peptide. We further show these disulfide bonds still form even when the initial affinity of the TCR-pMHC interaction is low. Accordingly, TCR-peptide disulfide bonds facilitate T cell activation by pMHC ligands with a wide spectrum of affinities for the TCR. Physiologically, this mechanism induces strong Zap70-dependent TCR signaling, which triggers T cell deletion or agonist selection in the thymus cortex. Covalent TCR-pMHC interactions may thus underlie a physiological T cell activation mechanism that has applications in basic immunology and potentially in immunotherapy.
Asunto(s)
Cisteína , Linfocitos T , Disulfuros , Antígenos de Histocompatibilidad , Complejo Mayor de Histocompatibilidad , Péptidos/química , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
The demonstration unit of the Universal Micromanipulation Robot (UMR) capable of semi-autonomous protein crystal harvesting has been tested and evaluated by independent users. We report the status and capabilities of the present unit scheduled for deployment in a high-throughput protein crystallization center. We discuss operational aspects as well as novel features such as micro-crystal handling and drip-cryoprotection, and we extrapolate towards the design of a fully autonomous, integrated system capable of reliable crystal harvesting. The positive to enthusiastic feedback from the participants in an evaluation workshop indicates that genuine demand exists and the effort and resources to develop autonomous protein crystal harvesting robotics are justified.
Asunto(s)
Automatización de Laboratorios/instrumentación , Cristalografía/instrumentación , Proteínas/química , Robótica , Cristalografía/métodos , Ergonomía , Humanos , MiniaturizaciónRESUMEN
Mannheimia haemolytica is the etiological agent of pneumonic pasteurellosis of cattle and sheep; two different OmpA subclasses, OmpA1 and OmpA2, are associated with bovine and ovine isolates, respectively. These proteins differ at the distal ends of four external loops, are involved in adherence, and are likely to play important roles in host adaptation. M. haemolytica is surrounded by a polysaccharide capsule, and the degree of OmpA surface exposure is unknown. To investigate surface exposure and immune specificity of OmpA among bovine and ovine M. haemolytica isolates, recombinant proteins representing the transmembrane domain of OmpA from a bovine serotype A1 isolate (rOmpA1) and an ovine serotype A2 isolate (rOmpA2) were overexpressed, purified, and used to generate anti-rOmpA1 and anti-rOmpA2 antibodies, respectively. Immunogold electron microscopy and immunofluorescence techniques demonstrated that OmpA1 and OmpA2 are surface exposed, and are not masked by the polysaccharide capsule, in a selection of M. haemolytica isolates of various serotypes and grown under different growth conditions. To explore epitope specificity, anti-rOmpA1 and anti-rOmpA2 antibodies were cross-absorbed with the heterologous isolate to remove cross-reacting antibodies. These cross-absorbed antibodies were highly specific and recognized only the OmpA protein of the homologous isolate in Western blot assays. A wider examination of the binding specificities of these antibodies for M. haemolytica isolates representing different OmpA subclasses revealed that cross-absorbed anti-rOmpA1 antibodies recognized OmpA1-type proteins but not OmpA2-type proteins; conversely, cross-absorbed anti-rOmpA2 antibodies recognized OmpA2-type proteins but not OmpA1-type proteins. Our results demonstrate that OmpA1 and OmpA2 are surface exposed and could potentially bind to different receptors in cattle and sheep.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Epítopos/metabolismo , Mannheimia haemolytica/clasificación , Pasteurelosis Neumónica/microbiología , Enfermedades de las Ovejas/microbiología , Animales , Anticuerpos Antibacterianos/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Bovinos , Epítopos/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Interacciones Huésped-Patógeno , Mannheimia haemolytica/inmunología , Mannheimia haemolytica/metabolismo , Ovinos , Especificidad de la EspecieRESUMEN
The data currently available on how the immune system recognises the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus; however, we lack data on how T cells are able to recognise this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.
Asunto(s)
COVID-19/inmunología , COVID-19/virología , Epítopos de Linfocito T/química , Antígeno HLA-A2/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Linfocitos T CD8-positivos/citología , Cristalografía por Rayos X , Citocinas/metabolismo , Epítopos/química , Antígeno HLA-A2/química , Humanos , Mutación , Péptidos/química , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Resonancia por Plasmón de Superficie , Linfocitos T Citotóxicos/inmunologíaRESUMEN
CD8+ T cells are crucial for anti-viral immunity; however, understanding T cell responses requires the identification of epitopes presented by human leukocyte antigens (HLA). To date, few SARS-CoV-2-specific CD8+ T cell epitopes have been described. Internal viral proteins are typically more conserved than surface proteins and are often the target of CD8+ T cells. Therefore, we have characterized eight peptides derived from the internal SARS-CoV-2 nucleocapsid protein predicted to bind HLA-A∗02:01, the most common HLA molecule in the global population. We determined not all peptides could form a complex with HLA-A∗02:01, and the six crystal structures determined revealed that some peptides adopted a mobile conformation. We therefore provide a molecular understanding of SARS-CoV-2 CD8+ T cell epitopes. Furthermore, we show that there is limited pre-existing CD8+ T cell response toward these epitopes in unexposed individuals. Together, these data show that SARS-CoV-2 nucleocapsid might not contain potent epitopes restricted to HLA-A∗02:01.
RESUMEN
Kelch-related protein 1 (Krp1) is up-regulated in oncogene-transformed fibroblasts. The Kelch repeats interact directly with the actin-binding protein Lasp-1 in membrane ruffles at the tips of pseudopodia, where both proteins are necessary for pseudopodial elongation. Herein, we investigate the molecular basis for this interaction. Probing an array of overlapping decapeptides of Rattus norvegicus (Rat) Krp1 with recombinant Lasp-1 revealed two binding sites; one ((317)YDPMENECYLT(327)) precedes the first of five Kelch repeats, and the other ((563)TEVNDIWKYEDD(574)) is in the last of the five Kelch repeats. Mutational analysis established that both binding sites are necessary for Krp1-Lasp-1 interaction in vitro and function in vivo. The crystal structure of the C-terminal domain of rat Krp1 (amino acids 289-606) reveals that both binding sites are brought into close proximity by the formation of a novel six-bladed beta-propeller, where the first blade is not formed by a Kelch repeat.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Motoras Moleculares/química , Proteínas del Tejido Nervioso/metabolismo , Seudópodos/ultraestructura , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Cristalografía por Rayos X , Proteínas del Citoesqueleto , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/fisiología , Proteínas Motoras Moleculares/metabolismo , Proteínas Motoras Moleculares/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Unión Proteica , Conformación Proteica , Ratas , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The enzyme transketolase from the lactic acid bacterium Lactobacillus salivarius (subsp. salivarius UCC118) has been recombinantly expressed and purified using an Escherichia coli expression system. Purified transketolase from L. salivarius has been crystallized using the vapour-diffusion technique. The crystals belonged to the trigonal space group P3(2)21, with unit-cell parameters a=b=75.43, c=184.11 A, and showed diffraction to 2.3 A resolution.
Asunto(s)
Lactobacillus/enzimología , Transcetolasa/química , Cristalización , Cristalografía por Rayos X , Expresión Génica , Transcetolasa/genética , Transcetolasa/aislamiento & purificaciónRESUMEN
Exceptionally large crystals of posnjakite, Cu4SO4(OH)6(H2O), formed during corrosion of a Swagelock(tm) Snubber copper gasket within the MX1 beamline at the ANSTO-Melbourne, Australian Synchrotron. The crystal structure was solved using synchrotron radiation to R 1 = 0.029 and revealed a structure based upon [Cu4(OH)6(H2O)O] sheets, which contain Jahn-Teller-distorted Cu octa-hedra. The sulfate tetra-hedra are bonded to one side of the sheet via corner sharing and linked to successive sheets via extensive hydrogen bonds. The sulfate tetra-hedra are split and rotated, which enables additional hydrogen bonds.
RESUMEN
Galactose-binding lectin from champedak (Artocarpus integer) consists of two chains: alpha and beta (133 and 21 amino acids, respectively). It has been shown to recognize and bind to carbohydrates involved in IgA and C1 inhibitor molecules. The protein was purified and crystallized at 293 K. Crystals were observed in two space groups, P2(1) and P2(1)2(1)2, and diffracted to 1.65 and 2.6 A, respectively.
Asunto(s)
Artocarpus/química , Galectinas/química , Cristalización , Cristalografía por Rayos XRESUMEN
Intracellular lipid-binding proteins (iLBPs) of the fatty acid-binding protein (FABP) family of animals transport, mainly fatty acids or retinoids, are confined to the cytosol and have highly similar 3D structures. In contrast, nematodes possess fatty acid-binding proteins (nemFABPs) that are secreted into the perivitelline fluid surrounding their developing embryos. We report structures of As-p18, a nemFABP of the large intestinal roundworm Ascaris suum, with ligand bound, determined using X-ray crystallography and nuclear magnetic resonance spectroscopy. In common with other FABPs, As-p18 comprises a ten ß-strand barrel capped by two short α-helices, with the carboxylate head group of oleate tethered in the interior of the protein. However, As-p18 exhibits two distinctive longer loops amongst ß-strands not previously seen in a FABP. One of these is adjacent to the presumed ligand entry portal, so it may help to target the protein for efficient loading or unloading of ligand. The second, larger loop is at the opposite end of the molecule and has no equivalent in any iLBP structure yet determined. As-p18 preferentially binds a single 18-carbon fatty acid ligand in its central cavity but in an orientation that differs from iLBPs. The unusual structural features of nemFABPs may relate to resourcing of developing embryos of nematodes.
Asunto(s)
Ascaris suum/química , Proteínas de Unión a Ácidos Grasos/química , Proteínas del Helminto/química , Óvulo/química , Animales , Ascaris suum/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Proteínas del Helminto/metabolismo , Ligandos , Óvulo/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
A novel, pure, synthetic material is presented that promotes the repair of full-thickness skin wounds. The active component is tropoelastin and leverages its ability to promote new blood vessel formation and its cell recruiting properties to accelerate wound repair. Key to the technology is the use of a novel heat-based, stabilized form of human tropoelastin which allows for tunable resorption. This implantable material contributes a tailored insert that can be shaped to the wound bed, where it hydrates to form a conformable protein hydrogel. Significant benefits in the extent of wound healing, dermal repair, and regeneration of mature epithelium in healthy pigs are demonstrated. The implant is compatible with initial co-treatment with full- and split-thickness skin grafts. The implant's superiority to sterile bandaging, commercial hydrogel and dermal regeneration template products is shown. On this basis, a new concept for a prefabricated tissue repair material for point-of-care treatment of open wounds is provided.
Asunto(s)
Implantes Absorbibles , Dermis , Hidrogel de Polietilenoglicol-Dimetacrilato , Andamios del Tejido , Tropoelastina , Cicatrización de Heridas/efectos de los fármacos , Animales , Autoinjertos/trasplante , Vasos Sanguíneos/metabolismo , Dermis/lesiones , Dermis/metabolismo , Dermis/patología , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Ratones , Porcinos , Tropoelastina/química , Tropoelastina/farmacologíaRESUMEN
Streptococcus pneumoniae contains a large number of sugar-transport systems and the system responsible for raffinose uptake has recently been identified. The substrate-binding protein component of this system shares strong sequence homology with the multiple sugar metabolism substrate-binding protein MsmE from S. mutans and contains a lipoprotein-attachment site at cysteine residue 23. A truncated form (residues 24-419) of RafE from S. pneumoniae was cloned and overexpressed in Escherichia coli. Native and selenomethionine-labelled protein have been crystallized in the hexagonal space group P6(1)22. Diffraction data have been successfully phased to 2.90 angstroms using Se SAD data and model building is in progress.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Lipoproteínas/química , Lipoproteínas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Rafinosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Selenometionina/metabolismo , Streptococcus pneumoniae/metabolismo , Difracción de Rayos XRESUMEN
Using the UHV experimental endstation on the soft X-ray beamline at the Australian Synchrotron, lysozyme and proteinase K crystals have been exposed to a vacuum of 10-5â mbar, prior to flash-cooling in a bath of liquid nitrogen. Subsequent data collection on the MX2 beamline at the Australian Synchrotron demonstrated that, for lysozyme and proteinase K, it is possible to subject these mounted crystals to a vacuum pressure of 10-5â mbar without destroying the crystal lattice. Despite the lower data quality of the vacuum-pumped crystals compared with control crystals, it is demonstrated that the protein crystals can survive in a vacuum under suitable conditions.
RESUMEN
Chemokines direct leukocyte migration by activating intracellular signalling pathways through G-protein coupled chemokine receptors. However, they also bind to other surface proteins, including a group of molecules which we refer to as 'atypical' chemokine receptors. One such molecule is D6. D6 is structurally-related to other chemokine receptors, and binds specific pro-inflammatory chemokines with high affinity, but surprisingly, when expressed in heterologous cell lines, it is unable to transduce signals after chemokine engagement. Instead, by using the approaches outlined in this chapter, evidence has emerged that D6 acts as a chemokine scavenger which uses unique intracellular trafficking properties to continuously sequester extracellular chemokines into cells. It is envisaged that this suppresses inflammation in vivo by limiting pro-inflammatory chemokine bioavailability, and indeed, D6 deficient mice show exaggerated inflammatory responses to a variety of challenges. In addition to the in vitro functional studies, we also describe the methods we have used to express, purify and analyse large quantities of D6 protein. The unusually high stability of D6 and its broad subcellular distribution enables D6 to be expressed to very high levels in transfected cells, making it possible, at least in principal, to produce enough D6 to allow for purification of quantities suitable for crystallisation. This is a key step on the path towards generating a three-dimensional structure of the molecule. Thus, the protocols we outline have helped establish chemokine scavenging as a novel paradigm in chemokine biology, and may also ultimately provide unprecedented insight into the structure of D6 and other chemokine receptors.