نبذة مختصرة : Programa de Doctorat en Biotecnologia / Tesi realitzada a l'Institut de Biologia Molecular de Barcelona (IBMB-CSIC)
نبذة مختصرة : [eng] The emergence and spread of antibiotic resistance among bacteria has become an undisputed global problem and one of the greatest threats to public health in the 21st century. The widespread, excessive and uncontrolled use of antibiotics, not only for therapeutic purposes but also in agriculture and animal husbandry, has resulted in a steady and rapid increase in the number of strains resistant to the drugs used. In all living cells, ribonucleotide reductases (RNRs) are essential enzymes that constitute the only known de novo pathway of deoxyribonucleotide biosynthesis (dNTP biosynthesis) via the catalyzed reduction of ribonucleoside triphosphates (NTPs, such as ATP, CTP, GTP, and TTP) using radical chemistry, thereby forming the fundamental building blocks for DNA synthesis and repair. The transcriptional repressor NrdR controls the expression of RNR genes in most bacteria and in some archaea. Importantly, NrdR is missing in eukaryotes, and as it is found in antibiotic resistant pathogens such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Staphylococcus aureus, it can be considered as a biomedical target. NrdR inhibits transcription of RNRs genes by binding its Zn-finger domain (ZFD) to a palindromic repeat of 16bp DNA that conform the so-called NrdR-boxes, which are upstream of RNRs promoter regions. An ATP-cone domain (ACD) present in NrdR, sensitive to the changes in concentration of (-deoxy) ribonucleoside triphosphates (dNTPs and NTPs), allosterically regulates the Zn-finger activity of NrdR. Several hypotheses on RNRs genes regulation by NrdR have been proposed, and in May 2022 a combination of biochemical and cryo-EM structural studies suggested mechanism of action for Streptomyces coelicolor (sc-) NrdR. Such a mechanism involves an ATP-loaded dodecamer, which cannot bind to DNA, a dATP/ATP- loaded octamer, and a dATP/ATP-loaded tetramer bound to the sc-nrdRJ promoter, which represses transcription of the RNR operon. In this doctoral thesis, the crystal structure of Escherichia coli NrdR revealed key interactions whose mutations altered the multimerization. To test the functional roles played by the different residues, in vitro assays were carried out that showed in solution the WT NrdR dimer instability in the absence of nucleotides, and elution of different assemblies in the presence of AMP, ADP, ATP, and dATP. The same studies performed with fusion NrdR and designed mutants in which the multimerization interactions were altered, revealed the importance of residues Glu36, Glu42, Tyr131 at the ZFD, and of segment aa 132-149 from the ACD in NrdR oligomerization. The highest impact was noted for mutation Glu42Ala and the deletion of aa 132-149 segment. Thus, both the ZFD and the ACD are fundamental for oligomerization and essential for the protein function. In vivo studies performed with single-site mutants indicated that mutation E42A at the ZFD completely abolished NrdR ability to repress transcription of RNRs, while mutation to Ala of multimerization-sensitive residues Glu36 at the ZFD and Tyr131 at the ACD did not cause a decrease of the repression level. Therefore, amino acid Glu42 is pivotal for the repressive function of RNR. The abundance of the NrdR protein in bacteria and extrapolation of the results obtained for E. coli and S. coelicolor NrdR points to an ATP/dATP-orchestrated mechanism, in which the type of NrdR multimers change and coordinates the repression activity of the RNR operon.
[spa] Se han propuesto varias hipótesis sobre la regulación de genes las reductasas de ribonucleótidos (RNRs) por NrdR, y en mayo de 2022 una combinación de estudios bioquímicos y la determinación de la estructura por cryo-EM sugirió un mecanismo de acción para la NrdR de Streptomyces coelicolor (sc-). Tal mecanismo implica un dodecámero cargado de ATP, que no puede unirse al ADN, un octámero cargado de dATP/ATP, y un tetrámero cargado de dATP/ATP unido al promotor sc-nrdRJ, que reprime la transcripción de sc-RNR. En esta tesis doctoral, la estructura cristalina de Escherichia coli NrdR reveló interacciones clave cuyas mutaciones alteraron la multimerización. Para probar los roles funcionales desempeñados por los diferentes residuos, se realizaron ensayos in vitro que mostraron en solución la inestabilidad del dímero WT NrdR en ausencia de nucleótidos, y la elución de diferentes ensambles en presencia de AMP, ADP, ATP y dATP. Los mismos estudios realizados con fusión NrdR y mutantes diseñados en los que se alteraban las interacciones de multimerización revelaron la importancia de los residuos Glu36, Glu42, Tyr131 en el dominio Zn-finger (ZFD) y del segmento aa 132-149 del dominio del cono de ATP (ACD) en la oligomerización NrdR. La observación de que la mutación Glu42Ala y la deleción de un segmento 132-149 generaban mayor impacto indicaron que tanto el ZFD como el ACD son fundamentales para la oligomerización, y que son esenciales para la función de la proteína. Los estudios in vivo realizados con mutantes puntuales mostraron como la sustitución E42A en el ZFD abolió completamente la capacidad de NrdR para reprimir la transcripción de RNRs, mientras que la mutación de otros residuos sensibles a la multimerización, Glu36 en el ZFD y Tyr131 en el ACD no causó una disminución del nivel de represión. Por lo tanto, el aminoácido Glu42 es fundamental para la función represiva de RNR. La abundancia de la proteína NrdR en bacterias y la extrapolación de los resultados obtenidos para E. coli y S. coelicolor NrdR apunta a un mecanismo orquestado por ATP/dATP, por el que el tipo de multímeros de NrdR cambia, coordinándose la actividad de represión del operón RNR.
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