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IMPORTANCE: Type IV pili and type II secretion systems are members of the widespread type IV filament (T4F) superfamily of nanomachines that assemble dynamic and versatile surface fibers in archaea and bacteria. The assembly and retraction of T4 filaments with diverse surface properties and functions require the plasma membrane platform proteins of the GspF/PilC superfamily. Generally considered dimeric, platform proteins are thought to function as passive transmitters of the mechanical energy generated by the ATPase motor, to somehow promote insertion of pilin subunits into the nascent pilus fibers. Here, we generate and experimentally validate structural predictions that support the trimeric state of a platform protein PulF from a type II secretion system. The PulF trimers form selective proton or sodium channels which might energize pilus assembly using the membrane potential. The conservation of the channel sequence and structural features implies a common mechanism for all T4F assembly systems. We propose a model of the oligomeric PulF-PulE ATPase complex that provides an essential framework to investigate and understand the pilus assembly mechanism.
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Sistemas de Secreción Tipo II , Sistemas de Secreción Tipo II/metabolismo , Klebsiella , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Adenosina Trifosfatasas/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismoRESUMEN
The outermost surface layer of any virus is formed by either a capsid shell or envelope. Such layers have traditionally been thought of as immovable structures, but it is becoming apparent that they cannot be viewed exclusively as static architectures protecting the viral genome. A limited number of proteins on the virion surface must perform a multitude of functions in order to orchestrate the viral life cycle, and allostery can regulate their structures at multiple levels of organization, spanning individual molecules, protomers, large oligomeric assemblies, or entire viral surfaces. Here, we review recent contributions from the molecular simulation field to viral surface allostery, with a particular focus on the trimeric spike glycoprotein emerging from the coronavirus surface, and the icosahedral flaviviral envelope complex. As emerging viral pathogens continue to pose a global threat, an improved understanding of viral dynamics and allosteric regulation will prove crucial in developing novel therapeutic strategies.
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Cápside , Ensamble de Virus , Ensamble de Virus/fisiología , Cápside/metabolismo , Simulación por Computador , Proteínas de la Cápside , ViriónRESUMEN
The changing landscape of SARS-CoV-2 Spike protein is linked to the emergence of variants, immune-escape and reduced efficacy of the existing repertoire of anti-viral antibodies. The functional activity of neutralizing antibodies is linked to their quaternary changes occurring as a result of antibody-Spike trimer interactions. Here, we reveal the conformational dynamics and allosteric perturbations linked to binding of novel human antibodies and the viral Spike protein. We identified epitope hotspots, and associated changes in Spike dynamics that distinguish weak, moderate and strong neutralizing antibodies. We show the impact of mutations in Wuhan-Hu-1, Delta, and Omicron variants on differences in the antibody-induced conformational changes in Spike and illustrate how these render certain antibodies ineffective. Antibodies with similar binding affinities may induce destabilizing or stabilizing allosteric effects on Spike, with implications for neutralization efficacy. Our results provide mechanistic insights into the functional modes and synergistic behavior of human antibodies against COVID-19 and may assist in designing effective antiviral strategies.
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COVID-19 , SARS-CoV-2 , Humanos , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Pruebas de NeutralizaciónRESUMEN
Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.
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Infecciones Bacterianas , Poliglactina 910 , Humanos , Ratones , Animales , Poliglactina 910/uso terapéutico , Suturas , Inflamación/tratamiento farmacológico , Infección de la Herida Quirúrgica/tratamiento farmacológico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , PéptidosRESUMEN
There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.
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Lipopolisacáridos , Receptores Toll-Like , Animales , Ratones , Porcinos , Lipopolisacáridos/metabolismo , Receptores Toll-Like/metabolismo , Inflamación/patología , Péptidos/química , Péptido Hidrolasas , Receptores de Lipopolisacáridos/metabolismoRESUMEN
Bats are the reservoir for numerous human pathogens, including coronaviruses. Despite many coronaviruses having descended from bat ancestors, little is known about virus-host interactions and broader evolutionary history involving bats. Studies have largely focused on the zoonotic potential of coronaviruses with few infection experiments conducted in bat cells. To determine genetic changes derived from replication in bat cells and possibly identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cell line. Here, we observed extensive deletions within the spike and open reading frame 4 (ORF4) genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses, but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that did not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon, abrogating spike expression but maintaining infection in bat cells. After passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions among virus subpopulations. Spike-independent infection of human coronavirus 229E may provide an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and known cellular entry receptors. IMPORTANCE Many viruses, including coronaviruses, originated from bats. Yet, we know little about how these viruses switch between hosts and enter human populations. Coronaviruses have succeeded in establishing in humans at least five times, including endemic coronaviruses and the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In an approach to identify requirements for host switches, we established a bat cell line and adapted human coronavirus 229E viruses by serial passage. The resulting viruses lost their spike protein but maintained the ability to infect bat cells, but not human cells. Maintenance of 229E viruses in bat cells appears to be independent of a canonical spike receptor match, which in turn might facilitate cross-species transmission in bats.
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COVID-19 , Quirópteros , Coronavirus Humano 229E , Animales , Humanos , Filogenia , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/metabolismoRESUMEN
Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognized by Toll-like receptor 4, mediating proinflammatory effects. We previously reported that LPS directly interacts with SARS-CoV-2 spike (S) protein and enhances proinflammatory activities. Using native gel electrophoresis and hydrogen-deuterium exchange mass spectrometry, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the S protein. Molecular simulations validated by a microscale thermophoresis binding assay revealed that LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting a role as an intermediate in LPS transfer. Congruently, nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells is strongly boosted by S2. Using NF-κB reporter mice followed by bioimaging, a boosting effect was observed for both S1 and S2, with the former potentially facilitated by proteolysis. The Omicron S variant binds to LPS, but with reduced affinity and LPS boosting in vitro and in vivo. Taken together, the data provide a molecular mechanism by which S protein augments LPS-mediated hyperinflammation.
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COVID-19 , FN-kappa B , Humanos , Ratones , Animales , FN-kappa B/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus , Lipopolisacáridos , SARS-CoV-2/metabolismoRESUMEN
SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein-LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Colorantes , Humanos , Lipopolisacáridos/metabolismo , Péptidos/metabolismo , Agregado de Proteínas , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Trombina/metabolismoRESUMEN
The COVID-19 pandemic has prompted a rapid response in vaccine and drug development. Herein, we modeled a complete membrane-embedded SARS-CoV-2 spike glycoprotein and used molecular dynamics simulations with benzene probes designed to enhance discovery of cryptic pockets. This approach recapitulated lipid and host metabolite binding sites previously characterized by cryo-electron microscopy, revealing likely ligand entry routes, and uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop. A full representation of glycan moieties was essential to accurately describe pocket dynamics. A multi-conformational behavior of the 617-628 loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry experiments, supportive of opening and closing dynamics. The pocket is the site of multiple mutations associated with increased transmissibility found in SARS-CoV-2 variants of concern including Omicron. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.
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SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Benceno , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Unión Proteica , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Recent developments in the SARS-CoV-2 pandemic point to its inevitable transformation into an endemic disease, urging both refinement of diagnostics for emerging variants of concern (VOCs) and design of variant-specific drugs in addition to vaccine adjustments. Exploring the structure and dynamics of the SARS-CoV-2 Spike protein, we argue that the high-mutability characteristic of RNA viruses coupled with the remarkable flexibility and dynamics of viral proteins result in a substantial involvement of allosteric mechanisms. While allosteric effects of mutations should be considered in predictions and diagnostics of new VOCs, allosteric drugs advantageously avoid escape mutations via non-competitive inhibition originating from alternative distal locations. The exhaustive allosteric signaling and probing maps presented herein provide a comprehensive picture of allostery in the spike protein, making it possible to locate potential mutations that could work as new VOC "drivers" and to determine binding patches that may be targeted by newly developed allosteric drugs.
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Tratamiento Farmacológico de COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , Mutación , Pandemias , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.
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Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Mutación Missense/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Antivirales/sangre , Sitios de Unión/genética , COVID-19/virología , Glicosilación , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina A/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Manosa/metabolismo , Mutación Missense/genética , Oligosacáridos/metabolismo , Polisacáridos/metabolismo , Prolina/genética , Prolina/inmunología , Prolina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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COVID-19/genética , Conformación Proteica , SARS-CoV-2/ultraestructura , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Animales , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Chlorocebus aethiops , Glicosilación , Humanos , Simulación de Dinámica Molecular , Unión Proteica/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células VeroRESUMEN
A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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The spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface angiotensin-converting enzyme 2 (ACE2) receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat [HR]) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the prefusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Thus, protease docking sites flanking the S1/S2 cleavage site represent alternate allosteric hotspot targets for potential therapeutic development.
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Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sitio Alostérico , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Sitios de Unión , COVID-19/metabolismo , Humanos , Espectrometría de Masas/métodos , Simulación de Dinámica Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteolisis , Receptores Virales/química , Receptores Virales/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Internalización del VirusRESUMEN
The high mutation rate in retroviruses is one of the leading causes of drug resistance. In human immunodeficiency virus type-1 (HIV-1), synergistic mutations in its protease and the protease substrate - the Group-specific antigen (Gag) polyprotein - work together to confer drug resistance against protease inhibitors and compensate the mutations affecting viral fitness. Some Gag mutations can restore Gag-protease binding, yet most Gag-protease correlated mutations occur outside of the Gag cleavage site. To investigate the molecular basis for this, we now report multiscale modelling approaches to investigate various sequentially cleaved Gag products in the context of clinically relevant mutations that occur outside of the cleavage sites, including simulations of the largest Gag proteolytic product in its viral membrane-bound state. We found that some mutations, such as G123E and H219Q, involve direct interaction with cleavage site residues to influence their local environment, while certain mutations in the matrix domain lead to the enrichment of lipids important for Gag targeting and assembly. Collectively, our results reveal why non-cleavage site mutations have far-reaching implications outside of Gag proteolysis, with important consequences for drugging Gag maturation intermediates and tackling protease inhibitor resistance.
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There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of â¼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.
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COVID-19/complicaciones , Inflamación/etiología , Lipopolisacáridos/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Sitios de Unión , COVID-19/inmunología , COVID-19/virología , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/inmunología , Modelos Animales de Enfermedad , Infecciones por Bacterias Gramnegativas/complicaciones , Infecciones por Bacterias Gramnegativas/inmunología , Humanos , Técnicas In Vitro , Lípido A/química , Lípido A/inmunología , Lípido A/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Inmunológicos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Síndrome de Dificultad Respiratoria/etiología , Factores de Riesgo , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.
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Dicroismo Circular/métodos , Modelos Moleculares , Fragmentos de Péptidos/análisis , Trombina/análisis , Secuencia de Aminoácidos , Animales , Concentración de Iones de Hidrógeno , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Trombina/química , Trombina/genéticaRESUMEN
The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.
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Cardiolipinas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Regulación Alostérica , Sitios de Unión , Proteínas Portadoras/genética , Cloranfenicol/farmacología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complejos Multiproteicos/química , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.