Crystal Structure of DnaK Protein Complexed with Nucleotide Exchange Factor GrpE in DnaK Chaperone System

Background: The Hsp70 chaperone cycle mediates stress-denatured protein refolding. Results: We present the structure of a DnaK-GrpE complex containing the DnaK interdomain linker and substrate-binding domain. Conclusion: Interaction between the DnaK linker/lid regions and the GrpE N-terminal α-helix and disordered region are essential for function. Significance: The structure provides a framework for studies concerning interaction of full-length DnaK and GrpE. The conserved, ATP-dependent bacterial DnaK chaperones process client substrates with the aid of the co-chaperones DnaJ and GrpE. However, in the absence of structural information, how these proteins communicate with each other cannot be fully delineated. For the study reported here, we solved the crystal structure of a full-length Geobacillus kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the interdomain linker (acting as a pseudo-substrate), and the N-terminal nucleotide-binding and C-terminal substrate-binding domains at 4.1-Å resolution. Each complex contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substrate binding, processing, and release during the Hsp70 chaperone cycle.

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