RESUMEN
Bacterial toxin-antitoxin (TA) systems are composed of a deleterious toxin and its antagonistic antitoxin. They are widespread in bacterial genomes and mobile genetic elements, and their functions remain largely unknown. Some TA systems, known as TAC modules, include a cognate SecB-like chaperone that assists the antitoxin in toxin inhibition. Here, we have investigated the involvement of proteases in the activation cycle of the TAC system of the human pathogen Mycobacterium tuberculosis. We show that the deletion of endogenous AAA+ proteases significantly bypasses the need for a dedicated chaperone and identify the mycobacterial ClpXP1P2 complex as the main protease involved in TAC antitoxin degradation. In addition, we show that the ClpXP1P2 degron is located at the extreme C-terminal end of the chaperone addiction (ChAD) region of the antitoxin, demonstrating that ChAD functions as a hub for both chaperone binding and recognition by proteases.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteínas Bacterianas/genética , Endopeptidasa Clp/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Chaperonas Moleculares/genética , Mycobacterium tuberculosis/genética , Sistemas Toxina-Antitoxina/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , Endopeptidasa Clp/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Genoma Bacteriano , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis (Msm) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of MsmClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Endopeptidasa Clp/ultraestructura , Mycobacterium smegmatis/metabolismo , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Endopeptidasa Clp/metabolismo , Simulación del Acoplamiento Molecular , Estructura Cuaternaria de Proteína , ProteolisisRESUMEN
Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest.