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Conformational characterization of DnaK and its complexes by small-angle X-ray scattering.

DnaK, a member of the 70 kDa heat shock protein (hsp70) family, and its complexes with substrate proteins and nucleotides were characterized by small-angle X-ray scattering (SAXS) and size-exclusion chromatography (SEC) techniques. The SAXS data indicated that DnaK has a dumbbell-shaped structure with a maximum dimension (dmax) of 112 angstrom, which is consistent with the reported two major functional domains [Chappell et al. (1987) J. Biol. Chem. 268, 12730-12735; Flaherty et al. (1990) Nature 346, 623-628]. The data were best fit by a model in which the two domains either are connected by a short hinge region or are just in contact with each other. The radius of gyration (Rg) of DnaK was determined as 37.5 +/- 1.0 angstrom in the absence of nucleotide. Binding of ATP induces a conformational change in DnaK as reflected by the changes in its P(r) function and Kratky plot, the increases (1-2 angstrom) in both its radius of gyration (Rg) and its Stokes radius (Rs), and the increase in its dmax (5-10 angstrom ). SAXS and SEC-HPLC results indicate that the association state of DnaK is very sensitive to the buffer concentration and the presence of substrates, as well as the protein concentration. At high buffer and protein concentrations, DnaK dimerizes, resulting in an increase in its apparent Rg and dmax values. The addition of substrate (unfolded protein or ATP) results in a return to the Rg value of monomeric DnaK, due to the dissociation of DnaK multimers induced by the substrate binding and resultant conformational changes. The DnaK-substrate protein complex gives a smaller Rg than expected, suggesting that the substrate protein binds to a cavity or cleft on DnaK rather than the exterior of the chaperone. The Kratky plot of the Gdn.HCl-induced unfolding intermediate state of DnaK is consistent with a compact, molten globule-like conformation, as previously suggested based on CD, fluorescence, and SEC-HPLC results [Palleros et al. (I 993) Biochemistry 32, 4314-4321].