RESUMEN

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 , Proteolisis

RESUMEN

The pupylation of cellular proteins plays a crucial role in the degradation cascade via the Pup-Proteasome system (PPS). It is essential for the survival of Mycobacterium smegmatis under nutrient starvation and, as such, the activity of many components of the pathway is tightly regulated. Here, we show that Pup, like ubiquitin, can form polyPup chains primarily through K61 and that this form of Pup inhibits the ATPase-mediated turnover of pupylated substrates by the 20S proteasome. Similarly, the autopupylation of PafA (the sole Pup ligase found in mycobacteria) inhibits its own enzyme activity; hence, pupylation of PafA may act as a negative feedback mechanism to prevent substrate pupylation under specific cellular conditions.

Asunto(s)

Proteínas Bacterianas/metabolismo , Mycobacterium smegmatis/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Adenosina Trifosfatasas/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Lisina/química , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/genética , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo