RESUMEN
The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into 13 pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane, and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis, and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol, and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis, which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the production of anticancer clerodanes through future metabolic engineering efforts.
Asunto(s)
Diterpenos de Tipo Clerodano , Diterpenos , Plantas Medicinales , Scutellaria , Diterpenos de Tipo Clerodano/química , Diterpenos de Tipo Clerodano/metabolismo , Scutellaria/genética , Scutellaria/química , Scutellaria/metabolismo , Abietanos/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Plantas Medicinales/genética , Plantas Medicinales/metabolismoRESUMEN
Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the "closed" pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more "closed" conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1.
Asunto(s)
Farmacorresistencia Viral , Síndrome de Inmunodeficiencia Adquirida del Felino/tratamiento farmacológico , Virus de la Inmunodeficiencia Felina , Infecciones por Lentivirus/tratamiento farmacológico , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/fisiología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Secuencia de Aminoácidos , Animales , Gatos , Cristalografía por Rayos X , Farmacorresistencia Viral/genética , Síndrome de Inmunodeficiencia Adquirida del Felino/enzimología , Virus de la Inmunodeficiencia Felina/enzimología , Virus de la Inmunodeficiencia Felina/genética , Infecciones por Lentivirus/enzimología , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
HIV-1 integrase (IN) catalyzes viral DNA integration into the host genome and facilitates multifunctional steps including virus particle maturation. Competency of IN to form multimeric assemblies is functionally critical, presenting an approach for anti-HIV strategies. Multimerization of IN depends on interactions between the distinct subunit domains and among the flanking protomers. Here, we elucidate an overlooked docking cleft of IN core domain that anchors the N-terminal helix-turn-helix (HTH) motif in a highly preserved and functionally critical configuration. Crystallographic structure of IN core domain in complex with Fab specifically targeting this cleft reveals a steric overlap that would inhibit HTH-docking, C-terminal domain contacts, DNA binding, and subsequent multimerization. While Fab inhibits in vitro IN integration activity, in vivo it abolishes virus particle production by specifically associating with preprocessed IN within Gag-Pol and interfering with early cytosolic Gag/Gag-Pol assemblies. The HTH-docking cleft may offer a fresh hotspot for future anti-HIV intervention strategies.
Asunto(s)
Integrasa de VIH/química , Integrasa de VIH/metabolismo , VIH-1/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Integrasa de VIH/genética , VIH-1/química , Secuencias Hélice-Giro-Hélice , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , ARN Viral/metabolismoRESUMEN
Viruses use a strategy of high mutational rates to adapt to environmental and therapeutic pressures, circumventing the deleterious effects of random single-point mutations by coevolved compensatory mutations, which restore protein fold, function or interactions damaged by initial ones. This mechanism has been identified as contributing to drug resistance in the HIV-1 Gag polyprotein and especially its capsid proteolytic product, which forms the viral capsid core and plays multifaceted roles in the viral life cycle. Here, we determined the X-ray crystal structure of C-terminal domain of the feline immunodeficiency virus (FIV) capsid and through interspecies analysis elucidate the structural basis of co-evolutionarily and spatially correlated substitutions in capsid sequences, which when otherwise uncoupled and individually substituted into HIV-1 capsid impair virion assembly and infectivity. The ability to circumvent the deleterious effects of single amino acid substitutions by cooperative secondary substitutions allows mutational flexibility that may afford viruses an important survival advantage. The potential of such interspecies structural analysis for preempting viral resistance by identifying such alternative but functionally equivalent patterns is discussed.
Asunto(s)
Sustitución de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Evolución Molecular , Virus de la Inmunodeficiencia Felina/genética , Mutación Missense , Cristalografía por Rayos X , Virus de la Inmunodeficiencia Felina/fisiología , Modelos Moleculares , Conformación Proteica , Ensamble de Virus , Internalización del VirusRESUMEN
The strong association of APOBEC3 cytidine deaminases with somatic mutations leading to cancers accentuates the importance of their tight intracellular regulation to minimize cellular transformations. We reveal a novel allosteric regulatory mechanism of APOBEC3 enzymes showing that APOBEC3G and APOBEC3A coordination of a secondary zinc ion, reminiscent to ancestral deoxycytidylate deaminases, enhances deamination activity. Zinc binding is pinpointed to loop-3 which whilst highly variable harbors a catalytically essential and spatially conserved asparagine at its N-terminus. We suggest that loop-3 may play a general role in allosterically tuning the activity of zinc-dependent cytidine deaminase family members.
Asunto(s)
Citidina Desaminasa/metabolismo , Zinc/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Citidina Desaminasa/química , Citidina Desaminasa/genética , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de SecuenciaRESUMEN
Retroviral DNA integration into the host genome is mediated by nucleoprotein assemblies containing tetramers of viral integrase (IN). Whereas the fully active form of IN comprises a dimer of dimers, the molecular basis of IN multimerization has not been fully characterized. IN has consistently been crystallized in an analogous dimeric form in all crystallographic structures and experimental evidence as to the level of similarity between IN monomeric and dimeric conformations is missing because of the lack of IN monomeric structures. Here we identify Phe187 as a critical dimerization determinant of IN from feline immunodeficiency virus (FIV), a nonprimate lentivirus that causes AIDS in the natural host, and report, in addition to a canonical dimeric structure of the FIV IN core-domain, a monomeric structure revealing the preservation of the backbone structure between the two multimeric forms and suggest a role for Phe187 in "hinging" the flexible IN dimer.