RESUMO
CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals remains unclear. Herein, we reported the crystal structure of the human CLEC12A-C-type lectin-like domain (CTLD) and identified a unique "basic patch" site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at the cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.
Assuntos
Lectinas Tipo C , Receptores Mitogênicos , Ácido Úrico , Humanos , Gota/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/imunologia , Receptores Mitogênicos/química , Receptores Mitogênicos/imunologia , Ácido Úrico/química , Ácido Úrico/imunologia , Domínios Proteicos , Cristalografia por Raios X , Imagem Individual de Molécula , Linhagem CelularRESUMO
The pharmacological inhibition of IDO1 is considered an effective therapeutic approach for cancer treatment. However, the inadequate response of existing holo-IDO1 inhibitors and unclear biomarkers available in clinical practice limit the possibility of developing efficacious IDO1 inhibitors. In the current study, we aimed to elucidate the activity and mechanism of a potent 1H-pyrrole-2-carboxylic acid derivative (B37) targeting apo-IDO1 and to determine its role in tumor therapy. By competing with heme for binding to apo-IDO1, B37 potently inhibited IDO1 activity, with an IC50 of 22 pM assessed using a HeLa cell-based assay. The x-ray cocrystal structure of the inhibitor-enzyme complex showed that the B37-human IDO1 complex has strong hydrophobic interactions, which enhances its binding affinity, determined using isothermal titration calorimetry. Stronger noncovalent interactions, including π stacking and hydrogen bonds formed between B37 and apo-human IDO1, underlay the enthalpy-driven force for B37 for binding to the enzyme. These binding properties endowed B37 with potent antitumor efficacy, which was confirmed in a mouse colon cancer CT26 syngeneic model in BALB/c mice and in an azoxymethane/dextran sulfate sodium-induced colon carcinogenesis model in C57BL/6 mice by activating the host immune system. Moreover, the combination of B37 and anti-PD1 Ab synergistically inhibited tumor growth. These results suggested that B37 may serve as a unique candidate for apo-IDO1 inhibition-mediated tumor immunotherapy.
Assuntos
Imunoterapia , Indolamina-Pirrol 2,3,-Dioxigenase , Animais , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-ß1 precursor (pro-TGF-ß1), integrins bind to the prodomain, apply force, and release the TGF-ß growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVß6 binds pro-TGF-ß1 in an orientation biologically relevant for force-dependent release of TGF-ß from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-ß orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin ß-subunit. Simulations of force-dependent activation of TGF-ß demonstrate evolutionary specializations for force application through the TGF-ß prodomain and through the ß- and not α-subunit of the integrin.
Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Integrinas/química , Integrinas/metabolismo , Fator de Crescimento Transformador beta1/agonistas , Fator de Crescimento Transformador beta1/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Evolução Molecular , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Fator de Crescimento Transformador beta1/metabolismoRESUMO
D assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D'D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D'D3 forms the binding site for factor VIII. The crystal structure of monomeric D'D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D' with its TIL' and E' modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL' domain of D' distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.
Assuntos
Fator VIII/metabolismo , Multimerização Proteica , Fator de von Willebrand/química , Sítios de Ligação , Cristalografia por Raios X , Dissulfetos , Retículo Endoplasmático/química , Complexo de Golgi/química , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Biogênese de Organelas , Ligação Proteica , Domínios Proteicos , Fator de von Willebrand/metabolismoRESUMO
The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVß6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVß6 affinity, in contrast to ß1 integrins. In integrin opening, rearrangement at the interface between the ßI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the ßI domain, which greatly increases in affinity in the open conformation. The larger size of the ßI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVß6 on-rate for both pro-TGF-ß1 and fibronectin declined. The results suggest that the open conformation of αVß6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVß6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.
Assuntos
Antígenos de Neoplasias/metabolismo , Citoesqueleto/metabolismo , Integrinas/metabolismo , Conformação Proteica , Fator de Crescimento Transformador beta1/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Sítios de Ligação , Humanos , Integrinas/química , Integrinas/genética , Ligantes , Manganês/metabolismo , Modelos Moleculares , Ligação Proteica , Deleção de Sequência , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta1/genéticaRESUMO
Integrins αVß6 and αVß8 are specialized for recognizing pro-TGF-ß and activating its growth factor by releasing it from the latency imposed by its surrounding prodomain. The integrin αVß8 is atypical among integrins in lacking sites in its cytoplasmic domain for binding to actin cytoskeleton adaptors. Here, we examine αVß8 for atypical binding to pro-TGF-ß1. In contrast to αVß6, αVß8 has a constitutive extended-closed conformation, and binding to pro-TGF-ß1 does not stabilize the open conformation of its headpiece. Although Mn2+ potently activates other integrins and increases affinity of αVß6 for pro-TGF-ß1 25- to 55-fold, it increases αVß8 affinity only 2- to 3-fold. This minimal effect correlates with the inability of Mn2+ and pro-TGF-ß1 to stabilize the open conformation of the αVß8 headpiece. Moreover, αVß8 was inhibited by high concentrations of Mn2+ and was stimulated and inhibited at markedly different Ca2+ concentrations than αVß6 These unusual characteristics are likely to be important in the still incompletely understood physiologic mechanisms that regulate αVß8 binding to and activation of pro-TGF-ß.
Assuntos
Integrinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Cálcio/metabolismo , Humanos , Manganês/metabolismoRESUMO
TGF-ß is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-ß is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked before proprotein convertase cleavage and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-ß1 with the proprotein convertase cleavage site mutated to mimic the structure of the TGF-ß1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure (i.e. the prodomain arm domain and GF domain in each monomer are swapped). Swapping has important implications for the mechanism of biosynthesis in the TGF-ß family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-ß family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain-GF complex are highly structurally conserved and which are perturbed by crystal lattice contacts.
Assuntos
Modelos Moleculares , Precursores de Proteínas/química , Fator de Crescimento Transformador beta1/química , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Mutação , Domínios Proteicos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα ß-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.
Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/química , Fator de von Willebrand/química , Sítios de Ligação , Cristalografia por Raios X , Evolução Molecular Direcionada , Humanos , Mutação , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Estrutura Quaternária de Proteína , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
While Interleukin 2 (IL2) has the capability to activate both NK and T cells robustly, its limited in vivo half-life, considerable toxicity, and tendency to boost Treg cells pose significant challenges, restricting its widespread application in cancer therapy. In this investigation, we engineered a novel IL2 variant (IL2-4M-PEG) with reduced CD25 binding activity and an extended half-life by substituting amino acids associated with CD25 binding and implementing site-directed PEGylation. IL2-4M-PEG notably amplifies effector cells over Treg cells. Furthermore, our findings reveal that IL2-4M-PEG, characterized by an extended half-life, exhibits anti-tumor effects in a mouse model. Consequently, this innovative IL2 holds the potential for enhancing combined cancer therapies in the future.
Assuntos
Imunoterapia , Subunidade alfa de Receptor de Interleucina-2 , Interleucina-2 , Polietilenoglicóis , Animais , Interleucina-2/metabolismo , Polietilenoglicóis/química , Imunoterapia/métodos , Humanos , Camundongos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Linhagem Celular Tumoral , Neoplasias/terapia , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Ligação Proteica , Camundongos Endogâmicos C57BL , Feminino , Camundongos Endogâmicos BALB C , Células Matadoras Naturais/imunologiaRESUMO
The proteolysis of VWF by ADAMTS13 is an essential step in the regulation of its hemostatic and thrombogenic potential. The cleavage occurs at strand ß4 in the structural core of the A2 domain of VWF, so unfolding of the A2 domain is a prerequisite for cleavage. In the present study, we present the crystal structure of an engineered A2 domain that exhibits a significant difference in the α3-ß4 loop compared with the previously reported structure of wild-type A2. Intriguingly, a metal ion was detected at a site formed mainly by the C-terminal region of the α3-ß4 loop that was later identified as Ca(²+) after various biophysical and biochemical studies. Force-probe molecular dynamic simulations of a modeled structure of the wild-type A2 featuring the discovered Ca(²+)-binding site revealed that an increase in force was needed to unfold strand ß4 when Ca(²+) was bound. Cleavage assays consistently demonstrated that Ca(²+) binding stabilized the A2 domain and impeded its unfolding, and consequently protected it from cleavage by ADAMTS13. We have revealed a novel Ca(²+)-binding site at the A2 domain of VWF and demonstrated a relationship between Ca(²+) and force in the regulation of VWF and primary hemostasis.
Assuntos
Proteínas ADAM/metabolismo , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Hemostasia/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteína ADAMTS13 , Sítios de Ligação/fisiologia , Biomarcadores Tumorais/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Mutagênese , Estrutura Terciária de Proteína , Desdobramento de Proteína , Trombose/metabolismo , Fatores de Transcrição/genéticaRESUMO
A cluster of several newly occurring mutations on Omicron is found at the ß-core region of the spike protein's receptor-binding domain (RBD), where mutation rarely happened before. Notably, the binding of SARS-CoV-2 to human receptor ACE2 via RBD happens in a dynamic airway environment, where mechanical force caused by coughing or sneezing occurs. Thus, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of RBDs and found that the mechanical stability of Omicron RBD increased by â¼20% compared with the wild type. Molecular dynamics (MD) simulations revealed that Omicron RBD showed more hydrogen bonds in the ß-core region due to the closing of the α-helical motif caused primarily by the S373P mutation. In addition to a higher unfolding force, we showed a higher dissociation force between Omicron RBD and ACE2. This work reveals the mechanically stabilizing effect of the conserved mutation S373P for Omicron and the possible evolution trend of the ß-core region of RBD.
RESUMO
The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
RESUMO
The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
Assuntos
Integrinas , Fibrose Pulmonar , Animais , Camundongos , Membrana Celular , Microscopia Crioeletrônica , Modelos Animais de DoençasRESUMO
Many questions about the significance of structural features of integrin α(V)ß(3) with respect to its mechanism of activation remain. We have determined and re-refined crystal structures of the α(V)ß(3) ectodomain linked to C-terminal coiled coils (α(V)ß(3)-AB) and four transmembrane (TM) residues in each subunit (α(V)ß(3)-1TM), respectively. The α(V) and ß(3) subunits with four and eight extracellular domains, respectively, are bent at knees between the integrin headpiece and lower legs, and the headpiece has the closed, low-affinity conformation. The structures differ in the occupancy of three metal-binding sites in the ßI domain. Occupancy appears to be related to the pH of crystallization, rather than to the physiologic regulation of ligand binding at the central, metal ion-dependent adhesion site. No electron density was observed for TM residues and much of the α(V) linker. α(V)ß(3)-AB and α(V)ß(3)-1TM demonstrate flexibility in the linker between their extracellular and TM domains, rather than the previously proposed rigid linkage. A previously postulated interface between the α(V) and ß(3) subunits at their knees was also not supported, because it lacks high-quality density, required rebuilding in α(V)ß(3)-1TM, and differed markedly between α(V)ß(3)-1TM and α(V)ß(3)-AB. Together with the variation in domain-domain orientation within their bent ectodomains between α(V)ß(3)-AB and α(V)ß(3)-1TM, these findings are compatible with the requirement for large structural changes, such as extension at the knees and headpiece opening, in conveying activation signals between the extracellular ligand-binding site and the cytoplasm.
Assuntos
Integrina alfaVbeta3/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cristalização , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Specific activation of amino acids by aminoacyl-tRNA synthetases is essential for maintaining translational fidelity. Here, we present crystal structures of Saccharomyces cerevisiae tryptophanyl-tRNA synthetase (sTrpRS) in apo form and in complexes with various ligands. In each complex, there is a sulfate ion bound at the active site which mimics the alpha- or beta-phosphate group of ATP during tryptophan activation. In particular, in one monomer of the sTrpRS-TrpNH(2)O complex, the sulfate ion appears to capture a snapshot of the alpha-phosphate of ATP during its movement towards tryptophan. Simulation study of a human TrpRS-Trp-ATP model shows that during the catalytic process the alpha-phosphate of ATP is driven to an intermediate position equivalent to that of the sulfate ion, then moves further and eventually fluctuates at around 2 A from the nucleophile. A conserved Arg may interact with the oxygen in the scissile bond at the transition state, indicating its critical role in the nucleophilic substitution. Taken together, eukaryotic TrpRSs may adopt an associative mechanism for tryptophan activation in contrast to a dissociative mechanism proposed for bacterial TrpRSs. In addition, structural analysis of the apo sTrpRS reveals a unique feature of fungal TrpRSs, which could be exploited in rational antifungal drug design.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Triptofano-tRNA Ligase/química , Triptofano/química , Monofosfato de Adenosina/química , Sequência de Aminoácidos , Antifúngicos/química , Apoenzimas/química , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/metabolismo , Sulfatos/química , Aminoacilação de RNA de Transferência , Triptofano/metabolismo , Triptofano-tRNA Ligase/metabolismoRESUMO
The ancient and ubiquitous aminoacyl-tRNA synthetases constitute a valuable model system for studying early evolutionary events. So far, the evolutionary relationship of tryptophanyl- and tyrosyl-tRNA synthetase (TrpRS and TyrRS) remains controversial. As TrpRS and TyrRS share low sequence homology but high structural similarity, a structure-based method would be advantageous for phylogenetic analysis of the enzymes. Here, we present the first crystal structure of an archaeal TrpRS, the structure of Pyrococcus horikoshii TrpRS (pTrpRS) in complex with tryptophanyl-5' AMP (TrpAMP) at 3.0 A resolution which demonstrates more similarities to its eukaryotic counterparts. With the pTrpRS structure, we perform a more complete structure-based phylogenetic study of TrpRS and TyrRS, which for the first time includes representatives from all three domains of life. Individually, each enzyme shows a similar evolutionary profile as observed in the sequence-based phylogenetic studies. However, TyrRSs from Archaea/Eucarya cluster with TrpRSs rather than their bacterial counterparts, and the root of TrpRS locates in the archaeal branch of TyrRS, indicating the archaeal origin of TrpRS. Moreover, the short distance between TrpRS and archaeal TyrRS and that between bacterial and archaeal TrpRS, together with the wide distribution of TrpRS, suggest that the emergence of TrpRS and subsequent acquisition by Bacteria occurred at early stages of evolution.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/classificação , Pyrococcus horikoshii/enzimologia , Triptofano-tRNA Ligase/química , Triptofano-tRNA Ligase/classificação , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Cristalografia por Raios X , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Triptofano/análogos & derivados , Triptofano/química , Triptofano-tRNA Ligase/genética , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/classificação , Tirosina-tRNA Ligase/genéticaRESUMO
Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 °C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.
Assuntos
Extremófilos , Proteínas do Envelope Viral , Extremófilos/metabolismo , Células Germinativas/metabolismo , Conformação Proteica , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismoRESUMO
The waning humoral immunity and emerging contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants resulted in the necessity of the booster vaccination of coronavirus disease 2019 (COVID-19). The inactivated vaccine, CoronaVac, is the most widely supplied COVID-19 vaccine globally. Whether the CoronaVac booster elicited adaptive responses that cross-recognize SARS-CoV-2 variants of concern (VoCs) among 77 healthy subjects receiving the third dose of CoronaVac were explored. After the boost, remarkable elevated spike-specific IgG and IgA responses, as well as boosted neutralization activities, were observed, despite 3.0-fold and 5.9-fold reduced neutralization activities against Delta and Omicron strains compared to that of the ancestral strain. Furthermore, the booster dose induced potent B cells and memory B cells that cross-bound receptor-binding domain (RBD) proteins derived from VoCs, while Delta and Omicron RBD-specific memory B cell recognitions were reduced by 2.7-fold and 4.2-fold compared to that of ancestral strain, respectively. Consistently, spike-specific circulating follicular helper T cells (cTfh) significantly increased and remained stable after the boost, with a predominant expansion towards cTfh17 subpopulations. Moreover, SARS-CoV-2-specific CD4+ and CD8+ T cells peaked and sustained after the booster. Notably, CD4+ and CD8+ T cell recognition of VoC spike was largely preserved compared to the ancestral strain. Individuals without generating Delta or Omicron neutralization activities had comparable levels of CD4+ and CD8+ T cells responses as those with detectable neutralizing activities. Our study demonstrated that the CoronaVac booster induced broad and potent adaptive immune responses that could be effective in controlling SARS-CoV-2 Delta and Omicron variants.