Based on the sequence homology, we have modeled the three-dimensional structure of Bacillus licheniformis DnaK (BlDnaK), a counterpart of Hsp70, and identified five different amino acids that might be responsible for maintaining ADP-Mg2+-Pi in the correct configuration at the ATP-binding cleft of the protein. As compared with wild-type BlDnaK, site-directed mutant proteins D8A, N13D, E145A, D168A, and T173A had a dramatic reduction in their chaperone activities. Complementation test revealed that the mutant proteins lost completely the ability to rescue the temperature-sensitive growth defect of Escherichia colidnaK756-ts. Wild-type BlDnak assisted the refolding of denatured firefly luciferase, whereas a significant decrease in this ability was observed for the mutant proteins. Simultaneous addition of B. licheniformis DnaJ, BlGrpE, and NR-peptide, did not synergistically stimulate the ATPase activity of D8A, E145A, D168A and T173A. Circular dichroism spectra were nearly identical for wild-type and mutant proteins, and they, except D8A, also exhibited a similar sensitivity towards temperature-induced denaturation. These results suggest that the selected residues are critical for the proper function of BlDnaK.
To elucidate the role of leucine 134 of Bacillus licheniformis nucleotide exchange factor (BlGrpE), site-saturation mutagenesis was employed to generate all possible replacements for this residue. Wild-type and mutant proteins were purified by nickel-chelated chromatography and had a molecular mass of approximately 34.5 kDa. As compared with wild-type BlGrpE, the nucleotide exchange factor (NEF) activity of L134H, L134K, L134R, L134D, L134E, L134N, L134Q, L134S, L134G and L134P was reduced by more than 96%. In vitro binding assay revealed that wild-type BlGrpE and the functional variants mainly interacted with the monomer of BlDnaK, but no such interaction was observed for the remaining mutant proteins. BlGrpE and 9 mutant proteins synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK), whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the alpha-helical content of the inactive mutant BlGrpEs was reduced significantly with respect to wild-type protein. Moreover, the inactive mutant proteins also exhibited a more sensitivity towards the temperature-induced denaturation. Taken together, these results indicate that Leu134 might play a structural role for the proper function of BlGrpE.
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