Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate

Abstract

DnaK is the canonical Hsp70 molecular chaperone protein from Escherichia coli. Like other Hsp70s, DnaK comprises two main domains: a 44-kDa N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, and a 25-kDa substrate-binding domain (SBD) that harbors the substrate-binding site. Here, we report an experimental structure for wild-type, full-length DnaK, complexed with the peptide NRLLLTG and with ADP. It was obtained in aqueous solution by using NMR residual dipolar coupling and spin labeling methods and is based on available crystal structures for the isolated NBD and SBD. By using dynamics methods, we determine that the NBD and SBD are loosely linked and can move in cones of +/-35 degrees with respect to each other. The linker region between the domains is a dynamic random coil. Nevertheless, an average structure can be defined. This structure places the SBD in close proximity of subdomain IA of the NBD and suggests that the SBD collides with the NBD at this area to establish allosteric communication.

Fig. 1.

Hybrid NMR RDC structure of E. coli DnaK. (Upper) Hybrid NMR RDC structure of E. coli (1–605) with ADP, orthophosphate, and the substrate peptide NRLLLTG bound. Red, NBD IA; green, NBD IB; blue, NBD IIA; yellow, NBD IIB; magenta, SBD BETA; cyan, SBD LID; black, NBD–SBD linker. The orientations and experimental uncertainties of the alignment tensors are shown in gray. (Lower) Hybrid NMR RDC structure of E. coli DnaK (1–605, blue) and WT E. coli DnaK (1–638, yellow) superposed on NBD IA, IB, and IIA. Both molecules have ADP, orthophosphate, and the substrate peptide NRLLLTG bound.

Fig. 2.

Results of MTSL spin labeling. (upper left) NH resonances that disappeared from the TROSY spectrum (orange spheres) when V210C (red spheres) is spin labeled with MTSL, indicated on E. coli DnaK with the correct SBD orientation. (upper right) NH resonances that disappeared from the TROSY spectrum (orange spheres) when V210C (red spheres) is spin labeled with MTSL, indicated on E. coli DnaK with the wrong SBD orientation. (lower left) NH within 25 Å (green spheres) of V210C (red spheres), indicated on E. coli DnaK with the correct SBD orientation. (lower right) NH within 25 Å (green spheres) of V210C (red spheres), indicated on E. coli DnaK with the wrong SBD orientation.

Fig. 3.

Mutagenesis and structure. The residues in E. coli DnaK, which when mutated affect the NBD–SBD interdomain communication, are represented as spheres in the colors of the subdomains to which they belong. These residues are, on the NBD: Y145A, N147A, and D148A (48); P143G and R151A (49); K155D and R167D (36). On the linker one has D393A (36). On the SBD they are K414I (31) and P419 (32, 33). Residues 417 and 418 SBD that show significant line broadening in the peptide-free form but not in the peptide bound form of the isolated SBD (11) are shown as dot surfaces.

Fig. 4.

Comparison of structures. Comparison of the Hsp coordinates as obtained for E. coli DnaK (1–605) with other published structures for multidomain constructs in the Hsp70 family. The Ca positions of the corresponding residues in the NBDs were superimposed. Dark blue, hybrid solution X-ray conformation of E. coli DnaK in the ADP/peptide state; yellow, T. thermophilus DnaK in the ADP/apo state (18); green, self-binding G. kaustophilus DnaK dimer in the ADP state (20); red, self-binding B. taurus Hsc70 (19); cyan, S. cerevisiae Hsp110 dimer in the ATP state (21).