RESUMO
Many neutralizing antibodies (nAbs) elicited to ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through natural infection and vaccination have reduced effectiveness to SARS-CoV-2 variants. Here, we show that therapeutic antibody ADG20 is able to neutralize SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize a broad range of VOCs, albeit with reduced potency against Omicron. Thus, this conserved and vulnerable site can be exploited for the design of universal vaccines and therapeutic antibodies.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Epitopos/imunologia , Humanos , Testes de Neutralização , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2'-phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat /KM ) of NADP(+) at least an order of magnitude lower than NAD(+) . Substitution of Glu42 with Ala improved specificity toward NADP(+) by 10-fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide-hydroxyl ion (NAD(+) -OH(-) ) adduct contraposing previously reported adducts. The OH(-) of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2'-hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD(+) -OH(-) adduct in proton relay instead of hydride transfer as noted for previous adducts.
Assuntos
Proteínas de Bactérias/química , Ácidos e Sais Biliares/biossíntese , Clostridium/enzimologia , Hidroxiesteroide Desidrogenases/química , Apoenzimas/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , NAD/químicaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas-specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure-based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family.
Assuntos
Proteínas de Bactérias/química , Pseudomonas aeruginosa , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Estrutura Secundária de Proteína , Percepção de QuorumRESUMO
Many neutralizing antibodies (nAbs) elicited to ancestral SARS-CoV-2 through natural infection and vaccination generally have reduced effectiveness to SARS-CoV-2 variants. Here we show therapeutic antibody ADG20 is able to neutralize all SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize all VOCs, albeit with reduced potency against Omicron. Thus, this highly conserved and vulnerable site can be exploited for design of universal vaccines and therapeutic antibodies.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Beta variant of concern (VOC) resists neutralization by major classes of antibodies from COVID-19 patients and vaccinated individuals. In this study, serum of Beta-infected patients revealed reduced cross-neutralization of wild-type virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of VOC-specific clonotypes and accommodation of mutations present in Beta and Omicron into a major antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with wild type-elicited antibodies, including a public VH1-58 clonotype that targets the RBD ridge. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift, with implications for design of next-generation vaccines and therapeutics.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Reações Cruzadas , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Deriva e Deslocamento Antigênicos , COVID-19/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 A resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic "whirly" single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.
Assuntos
Actinobacteria/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Divisão Celular , Microscopia Crioeletrônica , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Teste de Complementação Genética , Microscopia de Fluorescência/métodos , Microscopia de Contraste de Fase/métodos , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , Esporos BacterianosRESUMO
Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (â¼40-99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.
Assuntos
Proteínas de Bactérias/química , Regulação da Expressão Gênica , Neisseria gonorrhoeae/química , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Genoma Bacteriano , Modelos Moleculares , Dados de Sequência Molecular , Neisseria gonorrhoeae/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1â Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.
Assuntos
Aminoácidos/metabolismo , Bordetella bronchiseptica/enzimologia , Corismato Mutase/química , Dobramento de Proteína , Rhodobacteraceae/enzimologia , Sequência de Aminoácidos , Bacillus/enzimologia , Corismato Mutase/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
The crystal structure of Jann_2411 from Jannaschia sp. strain CCS1, a member of the Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45â Å by multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators.
Assuntos
Proteínas de Bactérias/química , Rhodobacteraceae/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Dedos de ZincoRESUMO
The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel α+ß fold comprising two ß-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.
Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/química , Lactobacillus plantarum/química , Dobramento de Proteína , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Lactobacillus plantarum/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
The crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.
Assuntos
Proteínas de Bactérias/química , Dobramento de Proteína , Rhodobacteraceae/química , Shewanella/química , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Genoma Bacteriano , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Shewanella/genética , Shewanella/metabolismo , Homologia Estrutural de ProteínaRESUMO
The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78â Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.
Assuntos
Bacillales/enzimologia , Pirofosfatases/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25â Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/ß SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the `open' conformation (YP_001095227.1), these helices are 15â Å apart, leading to the creation of a deep nonpolar cavity. In the `closed' structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their `open' monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.
Assuntos
Proteínas de Bactérias/química , Membrana Celular/química , Shewanella/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Shewanella/metabolismo , Homologia Estrutural de ProteínaRESUMO
KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.
Assuntos
Proteínas de Bactérias/química , Klebsiella pneumoniae/química , Lipoproteínas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a ß-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to ß-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins.
Assuntos
Bacteroides/química , Proteínas Periplásmicas/química , Sequência de Aminoácidos , Bacteroides/metabolismo , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Periplásmicas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
The crystal structure of the Bacteroides thetaiotaomicron protein BT_3984 was determined to a resolution of 1.7â Å and was the first structure to be determined from the extensive SusD family of polysaccharide-binding proteins. SusD is an essential component of the sus operon that defines the paradigm for glycan utilization in dominant members of the human gut microbiota. Structural analysis of BT_3984 revealed an N-terminal region containing several tetratricopeptide repeats (TPRs), while the signature C-terminal region is less structured and contains extensive loop regions. Sequence and structure analysis of BT_3984 suggests the presence of binding interfaces for other proteins from the polysaccharide-utilization complex.
Assuntos
Proteínas de Bactérias/química , Bacteroides/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.
Assuntos
Bacteroides/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Dobramento de Proteína , Sequência de Aminoácidos , Bacteroides/genética , Cristalografia por Raios X , Proteínas de Fímbrias/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05â Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a ß-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a ß-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal ß-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.
Assuntos
Proteínas de Bactérias/química , Bacteroides/química , Metabolismo dos Carboidratos , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Bacteroides/metabolismo , Sítios de Ligação , Carboidratos/química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.
Assuntos
Proteínas de Bactérias/química , Bacteroides/química , Perforina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)(+)-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD(+) has been determined to 2.0â Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/ß dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α-ß motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP(+) and NAD(+). The loop between ß5 and ß6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between ß5 and ß6) and Arg365' (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.