Substrate specificity and shuttling mechanism in DnaK-GroEL mediated cooperative protein folding

Cellular protein homeostasis is maintained by the molecular chaperones, where, DnaK/DnaJ/GrpE (KJE) and GroEL/GroES (ELS) chaperone systems play a central role by assisting protein folding, preventing aggregation and maintaining protein homeostasis. Despite the fundamental understanding on the activities and mechanistic roles of individual chaperone systems, the specificity, cooperativity and transfer mechanisms of substrates between chaperone networks still remain elusive. Here, using luciferase and malate dehydrogenase (MDH) as model substrates, we investigated the mechanistic details of substrate recognition and transfer, and the interplay among KJE and ELS chaperone systems. Our results show that the refolding efficiency of MDH is enhanced when both systems are present, indicating the synergistic effect of KJE and ELS. Interestingly, the refolding of KJE preferred substrate luciferase was inhibited by ELS, due to the formation of GroEL-luciferase complex as demonstrated by co-immunoprecipitation. Single-particle electron cryo-microscopy (cryo-EM) indicated the binding of luciferase with the apical domain of GroEL. Sequential chaperone addition experiments demonstrated ATP-dependent transfer of folding intermediates between chaperone systems, where, substrates initially engaged by one chaperone system can be effectively transferred and refolded by an ATP-driven complementary chaperone system. These findings uncover the dynamic and synergistic role of chaperone networks in processing various folding intermediates and protein conformers generated in response to stress and crowded cellular environment.