RESUMEN
Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR's research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the 1H,15N,13C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of ß-sheets corresponding to around 40% of the B2-3 backbone. The Cß chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy.
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The flavor profile of fermented fish products is influenced by the complex interplay of microbial and enzymatic actions on the raw materials. This review summarizes the various factors contributing to the unique taste and aroma of these traditional foods. Key ingredients include locally sourced fish species and a variety of spices and seasonings that enhance flavor while serving as cultural markers. Starter cultures also play a critical role in standardizing quality and accelerating fermentation. Flavor compounds in fermented fish are primarily derived from the metabolism of carbohydrates, lipids, and proteins, producing a diverse array of free amino acids, peptides, and volatile compounds such as aldehydes, ketones, alcohols, and esters. The fermentation process can be shortened by certain methods to reduce production time and costs, allowing for faster product turnover and increased profitability in the fermented fish market. Fermented fish products also show potent beneficial effects. This review highlights the importance of integrating traditional practices with modern scientific approaches. Future research directions to enhance the quality of fermented fish products are suggested.
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Influenza A virus poses a constant challenge to human health. The highly conserved influenza matrix-2 (M2) protein is an attractive target for the development of a universal antibody-based drug. However, screening using antigens with subphysiological conformation in a nonmembrane environment significantly reduces the generation of efficient antibodies. Here, M2(1-46) was incorporated into nanodiscs (M2-nanodiscs) with M2 in a membrane-embedded tetrameric conformation, closely resembling its natural physiological state in the influenza viral envelope. M2-nanodisc generation, an antigen, was followed by Chiloscyllium plagiosum immunization. The functional vNARs were selected by phage display panning strategy from the shark immune library. One of the isolated vNARs, AM2H10, could specifically bind to tetrameric M2 instead of monomeric M2e (the ectodomain of M2 protein). Furthermore, AM2H10 blocked ion influx through amantadine-sensitive and resistant M2 channels. Our findings indicated the possibility of developing functional shark nanobodies against various influenza viruses by targeting the M2 protein.
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Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.
Asunto(s)
Hialuronano Sintasas , Ácido Hialurónico , Phycodnaviridae , Microscopía por Crioelectrón , Hialuronano Sintasas/metabolismo , Phycodnaviridae/enzimología , PolímerosRESUMEN
A hallmark of Gram-negative bacteria is an asymmetric outer membrane containing lipopolysaccharides (LPSs) in the extracellular leaflet. LPS molecules consist of lipid A, which is connected to the inner and outer core oligosaccharides. This LPS core structure is extended in the periplasm by the O antigen, a variable and serotype-defining polysaccharide. In the ABC transporter-dependent LPS biosynthesis pathway, the WzmWzt transporter secretes the complete O antigen across the inner membrane for ligation to the LPS core. In some O antigen transporters, the nucleotide-binding domain of Wzt is fused C-terminally to a carbohydrate-binding domain (CBD) that interacts with the O antigen chain. Here, we present the crystal structure of the Aquifex aeolicus CBD that reveals a conserved flat and a variable twisted jelly-roll surface. The CBD dimer is stabilized by mutual ß strand exchange. Microbial glycan array binding studies with the isolated CBD provide insights into its interaction with complex carbohydrates.
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Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Antígenos O/metabolismo , Aquifex/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Transporte Biológico , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Transporte de ProteínasRESUMEN
O-antigens are cell surface polysaccharides of many Gram-negative pathogens that aid in escaping innate immune responses. A widespread O-antigen biosynthesis mechanism involves the synthesis of the lipid-anchored polymer on the cytosolic face of the inner membrane, followed by transport to the periplasmic side where it is ligated to the lipid A core to complete a lipopolysaccharide molecule. In this pathway, transport to the periplasm is mediated by an ATP-binding cassette (ABC) transporter, called Wzm-Wzt. Here we present the crystal structure of the Wzm-Wzt homologue from Aquifex aeolicus in an open conformation. The transporter forms a transmembrane channel that is sufficiently wide to accommodate a linear polysaccharide. Its nucleotide-binding domain and a periplasmic extension form 'gate helices' at the cytosolic and periplasmic membrane interfaces that probably serve as substrate entry and exit points. Site-directed mutagenesis of the gates impairs in vivo O-antigen secretion in the Escherichia coli prototype. Combined with a closed structure of the isolated nucleotide-binding domains, our structural and functional analyses suggest a processive O-antigen translocation mechanism, which stands in contrast to the classical alternating access mechanism of ABC transporters.
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Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Antígenos O/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Escherichia coli/química , Hidrólisis , Modelos Moleculares , Polisacáridos/metabolismo , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.
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Dominio Catalítico , Hialuronano Sintasas/metabolismo , Phycodnaviridae/enzimología , Proteínas Virales/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Molecular , Hialuronano Sintasas/química , Hialuronano Sintasas/genética , Ácido Hialurónico/biosíntesis , Multimerización de Proteína , Proteínas Virales/química , Proteínas Virales/genética , Xenopus laevisRESUMEN
Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).
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Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Animales , Biopolímeros/química , Biopolímeros/metabolismo , Quitina Sintasa/química , Quitina Sintasa/metabolismo , Humanos , Peso Molecular , Polisacáridos/química , Polisacáridos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Relación Estructura-ActividadRESUMEN
Store-operated Ca(2+) entry mediated by STIM1 and ORAI1 constitutes one of the major Ca(2+) entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca(2+) store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca(2+) signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.
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Canales de Calcio/metabolismo , Señalización del Calcio , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Cromatografía Liquida , Dicroismo Circular , Retículo Endoplásmico/metabolismo , Escherichia coli , Proteínas Fluorescentes Verdes , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes , Microscopía Confocal , Modelos Moleculares , Electroforesis en Gel de Poliacrilamida Nativa , Resonancia Magnética Nuclear Biomolecular , Proteína ORAI1 , Técnicas de Placa-Clamp , Conformación Proteica , Proteínas Recombinantes , Espectrometría de Fluorescencia , Molécula de Interacción Estromal 1 , Resonancia por Plasmón de Superficie , Proteína Fluorescente RojaRESUMEN
Phospholipid bilayer nanodiscs, a newly developed model membrane system, provides "native-like" membrane environment for membrane protein studies. Nanodiscs assembled by membrane scaffold protein and phospholipid bilayer, with defined sizes that can be accurately regulated by changing the amino acid residues of the MSP construct. Herein we described the expression and purification of ΔMSP, a deletion mutant of the membrane scaffold protein. Smaller nanodiscs with mixed lipids were assembled, and the observed (31)P NMR spectra showed identical chemical shifts to those of nanodiscs with pure POPC and POPE lipids, indicating they share similar chemical environments. The success of incorporation STIM1-TM into nanodiscs indicated the application of this smaller nanodisc system can be used to membrane protein studies by solution NMR.
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Membrana Dobles de Lípidos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Nanoestructuras/química , Resonancia Magnética Nuclear Biomolecular/métodos , Fosfolípidos , Secuencia de Aminoácidos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Fosfolípidos/química , Fosfolípidos/metabolismoRESUMEN
Dengue virus, belongs to Flaviviridae, is an arthropod transmitted virus that threatens millions of people's lives. As with other flaviviruses, a positive single-stranded 11-kilobases RNA in the dengue virus genome encodes three structural proteins (capsid protein C, membrane protein M, and envelope protein E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The two component protease NS2B-NS3p is essential for viral replication and is believed to be a potential antiviral drug target. Aprotinin, a native inhibitor, is proved to retard the activity of NS2B-NS3p. The backbone assignments of NS2B-NS3p will be essential for determining the high resolution solution structure of NS2B-NS3p and screening new antiviral drugs. Herein, we report the backbone (1)H, (15)N, (13)C resonance assignments of the N terminal fragment of NS2B (4.8 kDa) and NS3p (18.5 kDa) in complex with aprotinin (6.5 kDa) by high resolution NMR.
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Aprotinina/química , Virus del Dengue/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Aprotinina/metabolismo , Isótopos de Carbono , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Protones , ARN Helicasas/química , ARN Helicasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismoRESUMEN
Ionic protein-lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca(2+) can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor-CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively charged CD3ε/ζ cytoplasmic domains (CD3(CD)) and negatively charged phospholipids in the plasma membrane. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3(CD) dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca(2+) influx and TCR-proximal Ca(2+) concentration is higher than the average cytosolic Ca(2+) concentration. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca(2+) concentration induced the dissociation of CD3(CD) from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca(2+) influx. Moreover, when compared with wild-type cells, Ca(2+) channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca(2+) on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca(2+) with the non-physiological ion Sr(2+) resulted in the same feedback effect. Finally, (31)P NMR spectroscopy showed that Ca(2+) bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca(2+) has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca(2+) to T-cell activation involving direct lipid manipulation.
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Calcio/metabolismo , Activación de Linfocitos , Fosfolípidos/química , Fosfolípidos/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Animales , Calcio/farmacología , Membrana Celular/metabolismo , Citoplasma/metabolismo , Retroalimentación Fisiológica/efectos de los fármacos , Humanos , Células Jurkat , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Complejo Receptor-CD3 del Antígeno de Linfocito T/efectos de los fármacos , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Transducción de Señal/efectos de los fármacos , Solventes/química , Solventes/metabolismo , Electricidad Estática , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tirosina/metabolismoRESUMEN
Rhodanese domain is a ubiquitous structural module commonly found in bacterial, archaeal and eukaryotic cells. Growing evidence indicates that rhodanese domains act as the carrier of reactive sulfur atoms by forming persulfide intermediates in distinct metabolic pathways. YgaP, a membrane protein consisting of a rhodanese domain and a C-terminal transmembrane segment, is the only membrane-associated rhodanese in Escherichia coli. Herein, we report the resonance assignments of (1)H, (13)C and (15)N atoms of rhodanese domain of YgaP. Totally, chemical shifts of more than 95% of the atoms were assigned.
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Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Resonancia Magnética Nuclear Biomolecular , Tiosulfato Azufretransferasa/química , Secuencia de Aminoácidos , Isótopos de Carbono , Hidrógeno , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Estructura Terciaria de ProteínaRESUMEN
The mRNA degradation is an important regulatory mechanism which controls gene expression by limiting the number of translation times. Previous studies demonstrated that this process is essential for organisms. Escherichia coli RNA pyrophosphohydrolase (RppH) is an enzyme that triggers mRNA degradation by removing the 5' pyrophosphate, which is a rate-determining step. In order to understand the molecular mechanism of the biological function, the structural information of RppH is required. Herein, we report the resonance assignments of (1)H, (15)N, (13)C atoms of the E. coli RppH.