ClpB-mediated modulation of GroEL-bound luciferase and subsequent refolding by DnaK chaperone system

In prokaryotes, DnaK-DnaJ-GrpE (KJE), GroEL-GroES (ELS) and the AAA+ disaggregase ClpB constitute the major ATP-dependent chaperone systems involved in protein quality control machinery. Although the individual functions and interplay of KJE and ClpB systems have been extensively studied, the role of ClpB with ELS during KJE-mediated substrate refolding is still elusive. Here, we investigated the role of ClpB in KJE- and ELS-mediated refolding of chemically and heat-denatured luciferase. Simultaneous presence of KJE, ELS and ClpB significantly enhanced the refolding of chemically denatured luciferase, whereas the combination of KJE and ClpB resulted in a higher refolding yield of heat-denatured luciferase. The inhibitory effect of ELS on KJE-mediated refolding of luciferase was significantly reduced when KJE, ELS and ClpB were present simultaneously. Sequential chaperone addition experiments showed that ELS-bound heat-denatured luciferase was refolded to 24% and 36% of its native activity by KJE and ClpB respectively, whereas the KJE-ClpB chaperone system further enhanced refolding to 43%. ClpB alone did not enhanced the refolding of ELS-bound chemically denatured luciferase, while subsequent addition of KJE or KJE-ClpB restored 36% and 44% of the native activity, respectively. Turbidity assay showed that the KJE system effectively suppressed heat-induced luciferase aggregation, whereas ClpB alone exhibited no anti-aggregation activity. Collectively, these findings demonstrate that ClpB cooperates with the KJE chaperone system to enhance substrate refolding and rescue ELS-stalled substrate proteins, thus providing insights into the coordinated action of ATP-dependent chaperone systems in the maintenance of cellular protein homeostasis.