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 original version of this Article contained errors in Figures 1 and 4. In Fig. 1b, the Mtb-SecBTA sequence was displayed incorrectly. In the inset panel within Fig. 4c, the y-axis of the graph incorrectly read (Q.Rg)2 × I(Q)//(0), and should have read (Q.Rg)2 × I(Q)/I(0). These errors have been corrected in both the PDF and HTML versions of the Article.
RESUMEN
SecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis, we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB-SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Sistemas Toxina-Antitoxina/fisiología , Sitios de Unión , Chaperonas Moleculares/genética , Mycobacterium tuberculosis/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sistemas Toxina-Antitoxina/genéticaRESUMEN
SecB chaperones assist protein export in bacteria. However, certain SecB family members have diverged to become specialized toward the control of toxin-antitoxin (TA) systems known to promote bacterial adaptation to stress and persistence. In such tripartite TA-chaperone (TAC) systems, the chaperone was shown to assist folding and to prevent degradation of its cognate antitoxin, thus facilitating inhibition of the toxin. Here, we used both the export chaperone SecB of Escherichia coli and the tripartite TAC system of Mycobacterium tuberculosis as a model to investigate how generic chaperones can specialize toward the control of TA systems. Through directed evolution of SecB, we have identified and characterized mutations that specifically improve the ability of SecB to control our model TA system without affecting its function in protein export. Such a remarkable plasticity of SecB chaperone function suggests that its substrate binding surface can be readily remodeled to accommodate specific clients.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Chaperonas Moleculares/química , Mycobacterium tuberculosis/genética , Sistemas Toxina-Antitoxina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Evolución Molecular Dirigida , Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Mycobacterium tuberculosis/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Bacterial toxin-antitoxin (TA) systems, in which a labile antitoxin binds and inhibits the toxin, can promote adaptation and persistence by modulating bacterial growth in response to stress. Some atypical TA systems, known as tripartite toxin-antitoxin-chaperone (TAC) modules, include a molecular chaperone that facilitates folding and protects the antitoxin from degradation. Here we use a TAC module from Mycobacterium tuberculosis as a model to investigate the molecular mechanisms by which classical TAs can become 'chaperone-addicted'. The chaperone specifically binds the antitoxin at a short carboxy-terminal sequence (chaperone addiction sequence, ChAD) that is not present in chaperone-independent antitoxins. In the absence of chaperone, the ChAD sequence destabilizes the antitoxin, thus preventing toxin inhibition. Chaperone-ChAD pairs can be transferred to classical TA systems or to unrelated proteins and render them chaperone-dependent. This mechanism might be used to optimize the expression and folding of heterologous proteins in bacterial hosts for biotechnological or medical purposes.
