Fig. 2.

Identification of DnaK amino acid residues involved in Hsp90_Ec interaction in vivo. (a) Model of E. coli DnaK in the ADP-bound conformation [31] with the mutated residues used in this study shown as CPK models. The NBD is colored light orange and the SBD is gray. DnaK was rendered using PyMOL. (b, c) Interaction between DnaK wild-type or mutant and Hsp90_Ec in a bacterial two-hybrid system in vivo, as described in Materials and Methods. Interaction was measured by monitoring β-galactosidase activity on MacConkey indicator plates (b) and in liquid assays (c). In (b), a representative plate from three independent experiments is shown with colonies labeled 1 through 16. For colonies 2 through 13, all DnaK substitution mutants, named by substituted residues, have been constructed in T25-DnaK and are present in reactions with the T18-Hsp90_Ec construct. In (c), β-galactosidase activity is shown as mean ± SEM (n = 3) and are also presented in Supplemental Table S5.