RESUMEN
Rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals that are effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes, and potentially inhibit viruses sharing the same entry route. In this study, we report the identification of 1,3-diphenylurea (DPU) derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains. Importantly, the DPUDs did not induce any significant cytotoxicity at concentrations effective against the viral infections. Examining the uptake of cargoes specific to different endocytic pathways, we found that DPUDs majorly affected clathrin-mediated endocytosis, which both SARS-CoV-2 and IAV utilize for cellular entry. In the DPUD-treated cells, although virus binding on the cell surface was unaffected, internalization of both the viruses was drastically reduced. Since compounds similar to the DPUDs were previously reported to transport anions including chloride (Cl-) across lipid membrane and since intracellular Cl- concentration plays a critical role in regulating vesicular trafficking, we hypothesized that the observed defect in endocytosis by the DPUDs could be due to altered Cl- gradient across the cell membrane. Using in vitro assays we demonstrated that the DPUDs transported Cl- into the cell and led to intracellular Cl- accumulation, which possibly affected the endocytic machinery by perturbing intracellular Cl- homeostasis. Finally, we tested the DPUDs in mice challenged with IAV and mouse-adapted SARS-CoV-2 (MA 10). Treatment of the infected mice with the DPUDs led to remarkable body weight recovery, improved survival and significantly reduced lung viral load, highlighting their potential for development as broad-spectrum antivirals.
Asunto(s)
COVID-19 , Virus de la Influenza A , Animales , Ratones , SARS-CoV-2 , Virus de la Influenza A/fisiología , Endocitosis , Internalización del Virus , Antivirales/farmacología , Antivirales/químicaRESUMEN
The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.
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Antibacterianos , Staphylococcus aureus , Animales , Ratones , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Antibacterianos/farmacología , Girasa de ADN/química , Girasa de ADN/genética , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/farmacología , Péptidos/farmacologíaRESUMEN
Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 ß-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.
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COVID-19 , SARS-CoV-2 , Humanos , Mutación , Pliegue de Proteína , Proteínas , Supresión GenéticaRESUMEN
While many computational methods accurately predict destabilizing mutations, identifying stabilizing mutations has remained a challenge, because of their relative rarity. We tested ΔΔG0 predictions from computational predictors such as Rosetta, ThermoMPNN, RaSP, and DeepDDG, using 82 mutants of the bacterial toxin CcdB as a test case. On this dataset, the best computational predictor is ThermoMPNN, which identifies stabilizing mutations with a precision of 68%. However, the average increase in Tm for these predicted mutations was only 1°C for CcdB, and predictions were poorer for a more challenging target, influenza neuraminidase. Using data from multiple previously described yeast surface display libraries and in vitro thermal stability measurements, we trained logistic regression models to identify stabilizing mutations with a precision of 90% and an average increase in Tm of 3°C for CcdB. When such libraries contain a population of mutants with significantly enhanced binding relative to the corresponding wild type, there is no benefit in using computational predictors. It is then possible to predict stabilizing mutations without any training, simply by examining the distribution of mutational binding scores. This avoids laborious steps of in vitro expression, purification, and stability characterization. When this is not the case, combining data from computational predictors with high-throughput experimental binding data enhances the prediction of stabilizing mutations. However, this requires training on stability data measured in vitro with known stabilized mutants. It is thus feasible to predict stabilizing mutations rapidly and accurately for any system of interest that can be subjected to a binding selection or screen.
RESUMEN
IMPORTANCE: The Omicron subvariants have substantially evaded host-neutralizing antibodies and adopted an endosomal route of entry. The virus has acquired several mutations in the receptor binding domain and N-terminal domain of S1 subunit, but remarkably, also incorporated mutations in S2 which are fixed in Omicron sub-lineage. Here, we found that the mutations in the S2 subunit affect the structural and biological properties such as neutralization escape, entry route, fusogenicity, and protease requirement. In vivo, these mutations may have significant roles in tropism and replication. A detailed understanding of the effects of S2 mutations on Spike function, immune evasion, and viral entry would inform the vaccine design, as well as therapeutic interventions aiming to block the essential proteases for virus entry. Thus, our study has identified the crucial role of S2 mutations in stabilizing the Omicron spike and modulating neutralization resistance to antibodies targeting the S1 subunit.
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COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Endopeptidasas , Conformación Molecular , Mutación , Péptido Hidrolasas , SARS-CoV-2/clasificación , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Protein tertiary structure mimetics are valuable tools to target large protein-protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein-protein interaction through target dimerization.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Dimerización , Humanos , Péptidos/metabolismo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding. However, reliable screens for mutants that improve protein stability do not yet exist, especially for proteins that are well folded and relatively stable. Here, we demonstrate that incorporation of a single, specific, destabilizing mutation termed parent inactivating mutation into each member of a single-site saturation mutagenesis library, followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. We carried out fluorescence-activated cell sorting of such a yeast surface display, saturation suppressor library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations. We found that multiple stabilizing mutations could be identified after a single round of sorting. In addition, multiple libraries with different parent inactivating mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Finally, we show that individual stabilizing mutations could be combined to result in a multi-mutant that demonstrated an increase in thermal melting temperature of about 20 °C, and that displayed enhanced tolerance to high temperature exposure. We conclude that as this method is robust and employs small library sizes, it can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.
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Mutación Puntual , Estabilidad Proteica , Proteínas , Mutagénesis , Mutación Puntual/genética , Proteínas/química , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Single synonymous codon mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~ 200 single synonymous codon mutations in an operonic context by mutating almost all positions of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to most degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed. RESULTS: Synonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, predicted transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single synonymous codon mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate, but might also alter the folding kinetics of the protein in vivo. CONCLUSION: In summary, the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations.
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Proteínas de Escherichia coli , Escherichia coli , Biosíntesis de Proteínas , Mutación Silenciosa , Codón , Escherichia coli/genética , Fenotipo , Proteínas de Escherichia coli/genéticaRESUMEN
Deep mutational scanning studies suggest that synonymous mutations are typically silent and that most exposed, nonactive-site residues are tolerant to mutations. Here, we show that the ccdA antitoxin component of the Escherichia coli ccdAB toxin-antitoxin system is unusually sensitive to mutations when studied in the operonic context. A large fraction (â¼80%) of single-codon mutations, including many synonymous mutations in the ccdA gene shows inactive phenotype, but they retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure/stability, consistent with a large region of CcdA being intrinsically disordered. E. coli codon preference and strength of ribosome-binding associated with translation of downstream ccdB gene are found to be major contributors of the observed ccdA mutant phenotypes. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that the ccdA mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by studying single-site synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their operonic context, genes are likely to be more sensitive to both synonymous and nonsynonymous point mutations than inferred previously.
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Toxinas Bacterianas , Proteínas de Escherichia coli , Escherichia coli , Sistemas Toxina-Antitoxina , Proteínas Bacterianas , Toxinas Bacterianas/genética , Codón/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , MutaciónRESUMEN
Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128, and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1-infected elite neutralizer capable of neutralizing tier 3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Cisteína/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Vacunas contra el SIDA/inmunología , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular , Mapeo Epitopo/métodos , Células HEK293 , Infecciones por VIH/inmunología , Seropositividad para VIH/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Inmunización/métodos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The Mycobacterium tuberculosis genome harbors nine toxin-antitoxin (TA) systems that are members of the mazEF family, unlike other prokaryotes, which have only one or two. Although the overall tertiary folds of MazF toxins are predicted to be similar, it is unclear how they recognize structurally different RNAs and antitoxins with divergent sequence specificity. Here, we have expressed and purified the individual components and complex of the MazEF6 TA system from M. tuberculosis. Size exclusion chromatography-multiangle light scattering (SEC-MALS) was performed to determine the oligomerization status of the toxin, antitoxin, and the complex in different stoichiometric ratios. The relative stabilities of the proteins were determined by nano-differential scanning fluorimetry (nano-DSF). Microscale thermophoresis (MST) and yeast surface display (YSD) were performed to measure the relative affinities between the cognate toxin-antitoxin partners. The interaction between MazEF6 complexes and cognate promoter DNA was also studied using MST. Analysis of paired-end RNA sequencing data revealed that the overexpression of MazF6 resulted in differential expression of 323 transcripts in M. tuberculosis. Network analysis was performed to identify the nodes from the top-response network. The analysis of mRNA protection ratios resulted in identification of putative MazF6 cleavage site in its native host, M. tuberculosis. IMPORTANCE M. tuberculosis harbors a large number of type II toxin-antitoxin (TA) systems, the exact roles for most of which are unclear. Prior studies have reported that overexpression of several of these type II toxins inhibits bacterial growth and contributes to the formation of drug-tolerant populations in vitro. To obtain insights into M. tuberculosis MazEF6 type II TA system function, we determined stability, oligomeric states, and binding affinities of cognate partners with each other and with their promoter operator DNA. Using RNA-seq data obtained from M. tuberculosis overexpression strains, we have identified putative MazF6 cleavage sites and targets in its native, cellular context.
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Antitoxinas , Mycobacterium tuberculosis , Sistemas Toxina-Antitoxina , Tuberculosis , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas Bacterianas/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Sistemas Toxina-Antitoxina/genéticaRESUMEN
Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of â¼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
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Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Antivirales/biosíntesis , Vacunas contra la COVID-19/biosíntesis , COVID-19/prevención & control , Receptores Virales/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Animales , Anticuerpos Neutralizantes/biosíntesis , Sitios de Unión , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Cobayas , Células HEK293 , Calor , Humanos , Inmunogenicidad Vacunal , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Receptores Virales/química , Receptores Virales/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Vacunación , Potencia de la VacunaRESUMEN
HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens.IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.
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Sustitución de Aminoácidos , Ácido Aspártico/química , Proteína gp120 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , VIH-1/genética , Ácido Aspártico/metabolismo , Antígenos CD4/genética , Antígenos CD4/metabolismo , Clonación Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Mutagénesis , Multimerización de Proteína , Estabilidad Proteica , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Mycobacterium tuberculosis possesses an unusually large representation of type II toxin-antitoxin (TA) systems, whose functions and targets are mostly unknown. To better understand the basis of their unique expansion and to probe putative functional similarities among these systems, here we computationally and experimentally investigated their sequence relationships. Bioinformatic and phylogenetic investigations revealed that 51 sequences of the VapBC toxin family group into paralogous sub-clusters. On the basis of conserved sequence fingerprints within paralogues, we predicted functional residues and residues at the putative TA interface that are useful to evaluate TA interactions. Substitution of these likely functional residues abolished the toxin's growth-inhibitory activity. Furthermore, conducting similarity searches in 101 mycobacterial and â¼4500 other prokaryotic genomes, we assessed the relative conservation of the M. tuberculosis TA systems and found that most TA orthologues are well-conserved among the members of the M. tuberculosis complex, which cause tuberculosis in animal hosts. We found that soil-inhabiting, free-living Actinobacteria also harbor as many as 12 TA pairs. Finally, we identified five novel putative TA modules in M. tuberculosis. For one of them, we demonstrate that overexpression of the putative toxin, Rv2514c, induces bacteriostasis and that co-expression of the cognate antitoxin Rv2515c restores bacterial growth. Taken together, our findings reveal that toxin sequences are more closely related than antitoxin sequences in M. tuberculosis Furthermore, the identification of additional TA systems reported here expands the known repertoire of TA systems in M. tuberculosis.
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Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Biología Computacional/métodos , Mycobacterium tuberculosis/metabolismo , Sistemas Toxina-Antitoxina/genética , Secuencia de Aminoácidos , Antitoxinas/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/genética , Filogenia , Células Procariotas/metabolismo , Alineación de SecuenciaRESUMEN
pH is an important factor that affects the protein structure, stability, and activity. Here, we probe the nature of the low-pH structural form of the homodimeric CcdB (controller of cell death B) protein. Characterization of CcdB protein at pH 4 and 300 K using circular dichroism spectroscopy, 8-anilino-1-naphthalene-sulphonate binding, and Trp solvation studies suggests that it forms a partially unfolded state with a dry core at equilibrium under these conditions. CcdB remains dimeric at pH 4 as shown by multiple techniques, such as size-exclusion chromatography coupled to multiangle light scattering, analytical ultracentrifugation, and electron paramagnetic resonance. Comparative analysis using two-dimensional 15N-1H heteronuclear single-quantum coherence NMR spectra of CcdB at pH 4 and 7 suggests that the pH 4 and native state have similar but nonidentical structures. Hydrogen-exchange-mass-spectrometry studies demonstrate that the pH 4 state has substantial but anisotropic changes in local stability with core regions close to the dimer interface showing lower protection but some other regions showing higher protection relative to pH 7.
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Proteínas Bacterianas/química , Desplegamiento Proteico , Anisotropía , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Temperature-sensitive (Ts) mutants are important tools for understanding the role of essential gene(s), but their molecular basis is not well understood. We use CcdB ( Controller of Cell Death protein B) as a model system to explore the effects of Ts mutations on protein stability, folding, and ligand binding. Previously isolated Ts CcdB mutants fall broadly into two categories, namely, buried site (<5% accessibility) and active site (involved in DNA gyrase binding). Several mutants from each category were characterized. It was found that buried-site Ts mutants had decreased stability and foldability, higher aggregation propensity, and, in most cases, reduced affinity for gyrase at both permissive and restrictive temperatures. In contrast, exposed, active-site Ts mutants of CcdB exhibited stability either higher than or similar to that of the wild type and weakened inhibition of DNA gyrase function and/or reduced affinity for gyrase at a higher temperature. At all temperatures, Ts mutations at exposed, active-site residues primarily decrease specific activity without affecting protein levels, while Ts mutations at most buried residues decrease both specific activity and protein levels. Ts phenotypes in both cases arise because total activity is decreased below the threshold required for survival at the restrictive temperature but remains above it at the permissive temperatures. For several mutants, Ts phenotypes were ameliorated upon overexpression of the trigger factor chaperone, suggesting that Ts phenotypes may result from mutational effects on in vivo protein folding rather than on protein stability. This study delineates the diverse factors that contribute to Ts phenotypes. These insights can facilitate rational design of Ts mutants.
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Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/química , Escherichia coli/genética , Modelos Moleculares , Mutación , Fenotipo , Agregado de Proteínas , Conformación Proteica , Estabilidad Proteica , Desplegamiento Proteico , TemperaturaRESUMEN
Protein minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as human immunodeficiency virus, type 1 (HIV-1), because it can help in focusing the immune response toward conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer-domain immunogen (ODEC) that bound CD4-binding site (CD4bs) ligands with 3-12 µm affinity and elicited a modest neutralizing antibody response in rabbits. In this study, we have optimized ODEC using consensus sequence design, cyclic permutation, and structure-guided mutations to generate a number of variants with improved yields, biophysical properties, stabilities, and affinities (KD of 10-50 nm) for various CD4bs targeting broadly neutralizing antibodies, including the germline-reverted version of the broadly neutralizing antibody VRC01. In contrast to ODEC, the optimized immunogens elicited high anti-gp120 titers in rabbits as early as 6 weeks post-immunization, before any gp120 boost was given. Following two gp120 boosts, sera collected at week 22 showed cross-clade neutralization of tier 1 HIV-1 viruses. Using a number of different prime/boost combinations, we have identified a cyclically permuted OD fragment as the best priming immunogen, and a trimeric, cyclically permuted gp120 as the most suitable boosting molecule among the tested immunogens. This study also provides insights into some of the biophysical correlates of improved immunogenicity.
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Vacunas contra el SIDA/inmunología , Antígenos CD4/inmunología , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/inmunología , VIH-1/química , Vacunas contra el SIDA/química , Vacunas contra el SIDA/uso terapéutico , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , Anticuerpos ampliamente neutralizantes , Antígenos CD4/química , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/genética , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Ligandos , Unión Proteica , ConejosRESUMEN
Structure prediction methods often generate a large number of models for a target sequence. Even if the correct fold for the target sequence is sampled in this dataset, it is difficult to distinguish it from other decoy structures. An attempt to solve this problem using experimental mutational sensitivity data for the CcdB protein was described previously by exploiting the correlation of residue depth with mutational sensitivity (r ~ 0.6). We now show that such a correlation extends to four other proteins with localized active sites, and for which saturation mutagenesis datasets exist. We also examine whether incorporation of predicted secondary structure information and the DOPE model quality assessment score, in addition to mutational sensitivity, improves the accuracy of model discrimination using a decoy dataset of 163 targets from CASP. Although most CASP models would have been subjected to model quality assessment prior to submission, we find that the DOPE score makes a substantial contribution to the observed improvement. We therefore also applied the approach to CcdB and four other proteins for which reliable experimental mutational data exist and observe that inclusion of experimental mutational data results in a small qualitative improvement in model discrimination relative to that seen with just the DOPE score. This is largely because of our limited ability to quantitatively predict effects of point mutations on in vivo protein activity. Further improvements in the methodology are required to facilitate improved utilization of single mutant data.
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Proteínas/química , Animales , Dominio Catalítico , Bases de Datos de Proteínas , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagénesis , Mutación , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas/genéticaRESUMEN
Trimeric HIV-1 envelope (Env) immunogens are attractive due to their ability to display quaternary epitopes targeted by broadly neutralizing antibodies (bNAbs) while obscuring unfavorable epitopes. Results from the RV144 trial highlighted the importance of vaccine-induced HIV-1 Env V1V2-directed antibodies, with key regions of the V2 loop as targets for vaccine-mediated protection. We recently reported that a trimeric JRFL-gp120 immunogen, generated by inserting an N-terminal trimerization domain in the V1 loop region of a cyclically permuted gp120 (cycP-gp120), induces neutralizing activity against multiple tier-2 HIV-1 isolates in guinea pigs in a DNA prime/protein boost approach. Here, we tested the immunogenicity of cycP-gp120 in a protein prime/boost approach in rabbits and as a booster immunization to DNA/modified vaccinia Ankara (MVA)-vaccinated rabbits and rhesus macaques. In rabbits, two cycP-gp120 protein immunizations induced 100-fold higher titers of high-avidity gp120-specific IgG than two gp120 immunizations, with four total gp120 immunizations being required to induce comparable titers. cycP-gp120 also induced markedly enhanced neutralizing activity against tier-1A and -1B HIV-1 isolates, substantially higher binding and breadth to gp70-V1V2 scaffolds derived from a multiclade panel of global HIV-1 isolates, and antibodies targeting key regions of the V2-loop region associated with reduced risk of infection in RV144. Similarly, boosting MVA- or DNA/MVA-primed rabbits or rhesus macaques with cycP-gp120 showed a robust expansion of gp70-V1V2-specific IgG, neutralization breadth to tier-1B HIV-1 isolates, and antibody-dependent cellular cytotoxicity activity. These results demonstrate that cycP-gp120 serves as a robust HIV Env immunogen that induces broad anti-V1V2 antibodies and promotes neutralization breadth against HIV-1.IMPORTANCE Recent focus in HIV-1 vaccine development has been the design of trimeric HIV-1 Env immunogens that closely resemble native HIV-1 Env, with a major goal being the induction of bNAbs. While the generation of bNAbs is considered a gold standard in vaccine-induced antibody responses, results from the RV144 trial showed that nonneutralizing antibodies directed toward the V1V2 loop of HIV-1 gp120, specifically the V2 loop region, were associated with decreased risk of infection, demonstrating the need for the development of Env immunogens that induce a broad anti-V1V2 antibody response. In this study, we show that a novel trimeric gp120 protein, cycP-gp120, generates high titers of high-avidity and broadly cross-reactive anti-V1V2 antibodies, a result not found in animals immunized with monomeric gp120. These results reveal the potential of cycP-gp120 as a vaccine candidate to induce antibodies associated with reduced risk of HIV-1 infection in humans.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Inmunización/métodos , Vacunas contra el SIDA/genética , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Reacciones Cruzadas/inmunología , Diseño de Fármacos , Epítopos/química , Epítopos/inmunología , Cobayas , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Inmunización Secundaria , Inmunogenicidad Vacunal , Inmunoglobulina G/sangre , Macaca mulatta , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.