RESUMEN
Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.
Asunto(s)
Compuestos Azo/metabolismo , Atrofia Muscular Espinal/genética , Pirimidinas/metabolismo , Precursores del ARN/genética , Empalme del ARN , Compuestos Azo/química , Compuestos Azo/uso terapéutico , Secuencia de Bases , Sitios de Unión/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Exones/genética , Cinética , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica Molecular , Estructura Molecular , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/metabolismo , Mutación , Fármacos Neuromusculares/química , Fármacos Neuromusculares/metabolismo , Fármacos Neuromusculares/uso terapéutico , Conformación de Ácido Nucleico , Pirimidinas/química , Pirimidinas/uso terapéutico , Precursores del ARN/química , Precursores del ARN/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Production of flagella is costly and subject to global multilayered regulation, which is reflected in the hierarchical control of flagellar production in many bacterial species. For Salmonella enterica serovar Typhimurium and its relatives, global regulation of flagellar production primarily occurs through the control of flhDC transcription and mRNA translation. In this study, the roles of the homologous multidrug resistance regulators MarA, SoxS, Rob, and RamA (constituting the mar-sox-rob regulon in S Typhimurium) in regulating flagellar gene expression were explored. Each of these regulators was found to inhibit flagellar gene expression, production of flagella, and motility. To different degrees, repression via these transcription factors occurred through direct interactions with the flhDC promoter, particularly for MarA and Rob. Additionally, SoxS repressed flagellar gene expression via a posttranscriptional pathway, reducing flhDC translation. The roles of these transcription factors in reducing motility in the presence of salicylic acid were also elucidated, adding a genetic regulatory element to the response of S Typhimurium to this well-characterized chemorepellent. Integration of flagellar gene expression into the mar-sox-rob regulon in S Typhimurium contrasts with findings for closely related species such as Escherichia coli, providing an example of plasticity in the mar-sox-rob regulon throughout the Enterobacteriaceae family.IMPORTANCE The mar-sox-rob regulon is a large and highly conserved stress response network in the Enterobacteriaceae family. Although it is well characterized in E. coli, the extent of this regulon in related species is unclear. Here, the control of costly flagellar gene expression is connected to the mar-sox-rob regulon of S Typhimurium, contrasting with the E. coli regulon model. These findings demonstrate the flexibility of the mar-sox-rob regulon to accommodate novel regulatory targets, and they provide evidence for its broader regulatory role within this family of diverse bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Salmonella typhimurium/genética , Transactivadores/genética , Factores de Transcripción/genética , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/efectos de los fármacos , Flagelos/genética , Flagelos/metabolismo , Movimiento/fisiología , Biosíntesis de Proteínas , Ácido Salicílico/farmacología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins.