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Mucosa-associated lymphoid tissue lymphoma-translocation protein 1 (MALT1) is an attractive target for the development of modulatory compounds in the treatment of lymphoma and other cancers. While the three-dimensional structure of MALT1 has been previously determined through X-ray analysis, its dynamic behaviour in solution has remained unexplored. We present here dynamic analyses of the apo MALT1 form along with the E549A mutation. This investigation used NMR 15N relaxation and NOE measurements between side-chain methyl groups. Our findings confirm that MALT1 exists as a monomer in solution, and demonstrate that the domains display semi-independent movements in relation to each other. Our dynamic study, covering multiple time scales, along with the assessment of conformational populations by Molecular Dynamic simulations, Alpha Fold modelling and PCA analysis, put the side chain of residue W580 in an inward position, shedding light at potential mechanisms underlying the allosteric regulation of this enzyme.
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Simulación de Dinámica Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Regulación Alostérica , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Humanos , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , MutaciónRESUMEN
Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.
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The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.
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Antígenos de Histocompatibilidad Clase I , Neoplasias , Ratones , Animales , Antígenos de Histocompatibilidad Clase I/metabolismo , Receptor Leucocitario Tipo Inmunoglobulina B1/metabolismo , Células Asesinas Naturales , Ligandos , Péptidos/metabolismo , Neoplasias/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
MOTIVATION: Many approaches in systems biology have been applied in drug repositioning due to the increased availability of the omics data and computational biology tools. Using a multi-omics integrated network, which contains information of various biological interactions, could offer a more comprehensive inspective and interpretation for the drug mechanism of action (MoA). RESULTS: We developed a computational pipeline for dissecting the hidden MoAs of drugs (Open MoA). Our pipeline computes confidence scores to edges that represent connections between genes/proteins in the integrated network. The interactions showing the highest confidence score could indicate potential drug targets and infer the underlying molecular MoAs. Open MoA was also validated by testing some well-established targets. Additionally, we applied Open MoA to reveal the MoA of a repositioned drug (JNK-IN-5A) that modulates the PKLR expression in HepG2 cells and found STAT1 is the key transcription factor. Overall, Open MoA represents a first-generation tool that could be utilized for predicting the potential MoA of repurposed drugs and dissecting de novo targets for developing effective treatments. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/XinmengLiao/Open_MoA.
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Biología Computacional , Programas Informáticos , Reposicionamiento de MedicamentosRESUMEN
The dengue protease NS2B/NS3pro has been reported to adopt either an 'open' or a 'closed' conformation. We have developed a conformational filter that combines NMR with MD simulations to identify conformational ensembles that dominate in solution. Experimental values derived from relaxation parameters for the backbone and methyl side chains were compared with the corresponding back-calculated relaxation parameters of different conformational ensembles obtained from free MD simulations. Our results demonstrate a high prevalence for the 'closed' conformational ensemble while the 'open' conformation is absent, indicating that the latter conformation is most probably due to crystal contacts. Conversely, conformational ensembles in which the positioning of the co-factor NS2B results in a 'partially' open conformation, previously described in both MD simulations and X-ray studies, were identified by our conformational filter. Altogether, we believe that our approach allows for unambiguous identification of true conformational ensembles, an essential step for reliable drug discovery.
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Dengue , Péptido Hidrolasas , Humanos , Serina Endopeptidasas/química , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas no Estructurales Virales/químicaRESUMEN
Introduction: Human cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive. Methods: We employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses. Results: Here, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs. Discussion: These findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.
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Infecciones por Citomegalovirus , Humanos , Antígenos de Histocompatibilidad Clase I/metabolismo , Citomegalovirus/metabolismo , Células Asesinas Naturales , Péptidos/química , Antígenos HLA-ERESUMEN
G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.
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ADN Helicasas , Gránulos de Estrés , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Concentración de Iones de Hidrógeno , Ubiquitina TiolesterasaRESUMEN
Nuclear magnetic resonance (NMR) spectroscopy has become a formidable tool for biochemistry and medicine. Although J-coupling carries essential structural information it may also limit the spectral resolution. Homonuclear decoupling remains a challenging problem. In this work, we introduce a new approach that uses a specific coupling value as prior knowledge, and the Hankel property of the exponential NMR signal to achieve broadband heteronuclear decoupling using the low-rank method. Our results on synthetic and realistic HMQC spectra demonstrate that the proposed method not only effectively enhances resolution by decoupling, but also maintains sensitivity and suppresses spectral artefacts. The approach can be combined with non-uniform sampling, which means that the resolution can be further improved without any extra acquisition time.
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A new deep neural network based on the WaveNet architecture (WNN) is presented, which is designed to grasp specific patterns in the NMR spectra. When trained at a fixed non-uniform sampling (NUS) schedule, the WNN benefits from pattern recognition of the corresponding point spread function (PSF) pattern produced by each spectral peak resulting in the highest quality and robust reconstruction of the NUS spectra as demonstrated in simulations and exemplified in this work on 2D 1H-15N correlation spectra of three representative globular proteins with different sizes: Ubiquitin (8.6 kDa), Azurin (14 kDa), and Malt1 (44 kDa). The pattern recognition by WNN is also demonstrated for successful virtual homo-decoupling in a 2D methyl 1H-13C - HMQC spectrum of MALT1. We demonstrate using WNN that prior knowledge about the NUS schedule, which so far was not been fully exploited, can be used for designing new powerful NMR processing techniques that surpass the existing algorithmic methods.
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Imagen por Resonancia Magnética , Redes Neurales de la Computación , Espectroscopía de Resonancia Magnética/métodos , Ubiquitina , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Although PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells and a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Tumor-infiltrating NK cells that express PD-1 were highly associated with the expression of CXCR6. Furthermore, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wild-type mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells. These data demonstrate that there may be a role for the PD-1/PD-L1 axis in tumor-infiltrating NK cells in vivo.
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Mucosa-associated lymphoid tissue protein 1 (MALT1) plays a key role in adaptive immune responses by modulating specific intracellular signalling pathways that control the development and proliferation of both T and B cells. Dysfunction of these pathways is coupled to the progress of highly aggressive lymphoma as well as to potential development of an array of different immune disorders. In contrast to other signalling mediators, MALT1 is not only activated through the formation of the CBM complex together with the proteins CARMA1 and Bcl10, but also by acting as a protease that cleaves multiple substrates to promote lymphocyte proliferation and survival via the NF-κB signalling pathway. Herein, we present the partial 1H, 13C Ile/Val/Leu-Methyl resonance assignment of the monomeric apo form of the paracaspase-IgL3 domain of human MALT1. Our results provide a solid ground for future elucidation of both the three-dimensional structure and the dynamics of MALT1, key for adequate development of inhibitors, and a thorough molecular understanding of its function(s).
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Caspasas , FN-kappa B , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasas/metabolismo , Humanos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/química , Resonancia Magnética Nuclear BiomolecularRESUMEN
Significant advances in mass-spectroscopy (MS) have made it possible to investigate the cellular immunopeptidome, a large collection of MHC-associated epitopes presented on the surface of healthy, stressed and infected cells. These approaches have hitherto allowed the unambiguous identification of large cohorts of epitope sequences that are restricted to specific MHC class I and II molecules, enhancing our understanding of the quantities, qualities and origins of these peptide populations. Most importantly these analyses provide essential information about the immunopeptidome in responses to pathogens, autoimmunity and cancer, and will hopefully allow for future tailored individual therapies. Protein post-translational modifications (PTM) play a key role in cellular functions, and are essential for both maintaining cellular homeostasis and increasing the diversity of the proteome. A significant proportion of proteins is post-translationally modified, and thus a deeper understanding of the importance of PTM epitopes in immunopeptidomes is essential for a thorough and stringent understanding of these peptide populations. The aim of the present review is to provide a structural insight into the impact of PTM peptides on stability of MHC/peptide complexes, and how these may alter/modulate immune responses.
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Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.
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Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Células Asesinas Naturales , Células Progenitoras Linfoides , Animales , Diferenciación Celular/inmunología , Proteína Forkhead Box O1/inmunología , Proteína Forkhead Box O3/inmunología , Inmunidad Innata , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Presentation of pathogen-derived epitopes by major histocompatibility complex I (MHC-I) can lead to the activation and expansion of specific CD8+ T cell clones, eventually resulting in the destruction of infected target cells. Altered peptide ligands (APLs), designed to elicit immunogenicity toward a wild-type peptide, may affect the overall stability of MHC-I/peptide (pMHC) complexes and modulate the recognition by T cell receptors (TCR). Previous works have demonstrated that proline substitution at position 3 (p3P) of different MHC-restricted epitopes, including the immunodominant LCMV-derived epitope gp33 and escape variants, may be an effective design strategy to increase epitope immunogenicity. These studies hypothesized that the p3P substitution increases peptide rigidity, facilitating TCR binding. Here, molecular dynamics simulations indicate that the p3P modification rigidifies the APLs in solution predisposing them for the MHC-I loading as well as once bound to H-2Db, predisposing them for TCR binding. Our results also indicate that peptide position 6, key for interaction of H-2Db/gp33 with the TCR P14, takes a suboptimal conformation before as well as after binding to the TCR. Analyses of H-2Db in complex with APLs, in which position 6 was subjected to an l- to d-amino acid modification, revealed small conformational changes and comparable pMHC thermal stability. However, the l- to d-modification reduced significantly the binding to P14 even in the presence of the p3P modification. Our combined data highlight the sensitivity of the TCR for the conformational dynamics of pMHC and provide further tools to dissect and modulate TCR binding and immunogenicity via APLs.
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The serotype II Dengue (DENV 2) virus is the most prevalent of all four known serotypes. Herein, we present nearly complete 1H, 15N, and 13C backbone and 1H, 13C isoleucine, valine, and leucine methyl resonance assignment of the apo S135A catalytically inactive variant of the DENV 2 protease enzyme folded as a tandem formed between the serine protease domain NS3pro and the cofactor NS2B, as well as the secondary structure prediction of this complex based on the assigned chemical shifts using the TALOS-N software. Our results provide a solid ground for future elucidation of the structure and dynamic of the apo NS3pro/NS2B complex, key for adequate development of inhibitors, and a thorough molecular understanding of their function(s).
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Virus del Dengue , Dengue , Virus del Dengue/química , Virus del Dengue/metabolismo , Humanos , Proteínas Mutantes , Resonancia Magnética Nuclear Biomolecular , Proteínas no Estructurales Virales/químicaRESUMEN
Autoantigen discovery is a critical challenge for the understanding and diagnosis of autoimmune diseases. While autoantibody markers in current clinical use have been identified through studies focused on individual disorders, we postulated that a reverse approach starting with a putative autoantigen to explore multiple disorders might hold promise. We here targeted the epidermal protein transglutaminase 1 (TGM1) as a member of a protein family prone to autoimmune attack. By screening sera from patients with various acquired skin disorders, we identified seropositive subjects with the blistering mucocutaneous disease paraneoplastic pemphigus. Validation in further subjects confirmed TGM1 autoantibodies as a 55% sensitive and 100% specific marker for paraneoplastic pemphigus. This gene-centric approach leverages the wealth of data available for human genes and may prove generally applicable for biomarker discovery in autoimmune diseases.
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Autoantígenos/sangre , Síndromes Paraneoplásicos/inmunología , Pénfigo/inmunología , Transglutaminasas/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Síndromes Paraneoplásicos/sangre , Pénfigo/sangre , Adulto JovenRESUMEN
Despite progress in the treatment of non-visceral malignancies, the prognosis remains poor for malignancies of visceral organs and novel therapeutic approaches are urgently required. We evaluated a novel therapeutic regimen based on treatment with Se-methylselenocysteine (MSC) and concomitant tumor-specific induction of Kynurenine aminotransferase 1 (KYAT1) in hepatocellular carcinoma (HCC) cell lines, using either vector-based and/or lipid nanoparticle-mediated delivery of mRNA. Supplementation of MSC in KYAT1 overexpressed cells resulted in significantly increased cytotoxicity, due to ROS formation, as compared to MSC alone. Furthermore, microRNA antisense-targeted sites for miR122, known to be widely expressed in normal hepatocytes while downregulated in hepatocellular carcinoma, were added to specifically limit cytotoxicity in HCC cells, thereby limiting the off-target effects. KYAT1 expression was significantly reduced in cells with high levels of miR122 supporting the concept of miR-guided induction of tumor-specific cytotoxicity. The addition of alpha-ketoacid favored the production of methylselenol, enhancing the cytotoxic efficacy of MSC in HCC cells, with no effects on primary human hepatocytes. Altogether, the proposed regimen offers great potential to safely and specifically target hepatic tumors that are currently untreatable.
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The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is down-regulated due to the low expression of MHC-class I (MHC-I) molecules on the cell's surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells.
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Antígenos de Histocompatibilidad Clase I , Neoplasias , Células Presentadoras de Antígenos , Análisis por Conglomerados , Colon , Antígenos de Histocompatibilidad Clase II , Complejo Mayor de HistocompatibilidadRESUMEN
SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities.
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Infecciones por Citomegalovirus/virología , Citomegalovirus/patogenicidad , Infecciones por Herpesviridae/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genética , Antivirales/farmacología , Quinasas Ciclina-Dependientes/metabolismo , Citomegalovirus/genética , Citoplasma/metabolismo , Citoplasma/virología , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosforilación , Replicación Viral/efectos de los fármacosRESUMEN
SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.