Crystal Structure of a Thermophilic GrpE Protein: Insight into Thermosensing Function for the DnaK Chaperone System

Abstract

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpE(Tth)) at 3.23 A resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpE(Eco)), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpE(Tth). In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpE(Eco) structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpE(Tth) correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpE(Tth) causes functional deficiency of nucleotide exchange factor in response to the heat shock.

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

“…Similarities and Differences among GrpE Structures-The overall structural features of GkGrpE are consistent with those of EcoGrpE (18) and TthGrpE (30) and include the long N-terminal ␣-helix, the central four-helix bundle, and the C-terminal ␤-sheet domain (Fig. 4A).…”

Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

“…This flexibility is found in two places. The first is the N-terminal region of the ␣-helix (residues 34 -68), whose plasticity was observed in the crystal structures of different GrpE molecules (16,17,20) and is clear in the three-dimensional reconstructions of the DnaKGrpE complexes we generated here. Bending of the N-terminal half of the helices, including residues 34 -68, allows this segment to interact with the linker (Fig.…”

“…The tail is formed by an N-terminal disordered region, and a long ␣-helix domain that forms a coiled-coil structure with the opposite monomer. The head is formed by a four-helix bundle with two helices from each monomer, followed by a compact ␤-sheet domain (15,16). The atomic structure of a complex between the GrpE dimer and DnaK NBD was determined several years ago (17) (Fig.…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…Besides DnaJ, several other co-chaperones and nucleotide exchange factors modulate DnaK, although the latter class of proteins has so far not been studied by NMR and only crystal structures of the of the nucleotide exchange factor GrpE in the free and the DnaK-bound form exist [163][164][165]. Mammalian Bag1 is a co-chaperone interacting with Hsc70 in an antagonistic way by suppressing refolding of substrate proteins [166].…”