FIGURE 2.

Covalently linked ClpB NBD1-M dimer variants with intermolecular disulfide bonds. A, schematic representation of a disulfide-linked NBD1-M dimer. The structural close-up view is based on the available crystal structure of ClpB (Protein Data Bank code 1QVR) and shows the catalytic site located in the interface between two NBD1 subunits (green and yellow, respectively) with the conserved, trans-acting arginines Arg-322 and Arg-323 in close proximity to the ATP molecule bound to the neighboring subunit. Lys-204 and Glu-271 are part of the catalytically essential Walker A and Walker B motifs, respectively. Two pairs of cysteines, P221C/M394C and Q184C/A390C, were introduced by site-directed mutagenesis to form intermolecular disulfide bonds, but only the first pair was used in further experiments. Different mutated dimer variants were designed, as shown in C–F. B, disaggregation activity of a full-length ClpB dimer cross-linked by the P221C/M394C disulfide bond. The relative α-glucosidase activity (normalized against the positive control) for different time points during the ClpB/DnaK/DnaJ/GrpE-assisted disaggregation reaction is shown for the unlinked ClpB wild-type protein (red) and the cross-linked ClpB dimer (blue). Negative controls were as follows: no chaperones present (black), only ClpB present (wild type (gray) and cross-linked dimer (yellow)), and only DnaK/DnaJ/GrpE present (brown). C, NBD1-M dimers with Walker A mutation K204Q either in the cross-linked interface or in the free interface. The mutation leads to a nucleotide binding-deficient catalytic site. D, NBD1-M dimers with Walker B mutation E271Q either in the cross-linked interface or in the free interface. The mutation leads to an ATP hydrolysis-deficient catalytic site that remains nucleotide binding-competent. E, NBD1-M dimers with R322A or R323A mutation in the cross-linked interface. F, NBD1-M dimers with R322A or R323A mutation in combination with Walker B mutation E271Q in the cross-linked interface.