Extended Data Fig. 2: Translocation by ClpB variants.
From: Processive extrusion of polypeptide loops by a Hsp100 disaggregase
a, ClpB monomer structure indicating all tested variants. These variants (except K467C) were generated in the constitutively active Y503D background. Variants E279A and E678A are Walker B mutants in the nucleotide-binding domains NBD1 and NBD2, respectively. These mutations abolish ATP hydrolysis at NBD1 or NBD2. Variants Y251A and Y653A are pore-loop mutants in NBD1 and NBD2, respectively. These mutations affect substrate interaction in the ClpB pore at either NBD1 or NBD2. The K476C variant undocks the middle domain (MD), mimicking the effect of Hsp70 (DnaK) activation. MD undocking in the Y503D variant is more pronounced, and therefore activation is more robust. An additional construct (ClpB(ΔN)) lacked the N-terminal domain (NTD), hindering initial substrate binding. Finally, the variant E731C harbours a cysteine at the bottom of NBD2 for fluorophore labelling. b, Fraction of time showing activity (fA) for different mutants (in Y503D background, except K476C and wild type (WT)). c, Average translocation speed for all ClpB variants tested. KJE is the DnaK system (DnaK, DnaJ and GrpE). The median is displayed as a horizontal line within the box, and the mean as a white square. Whiskers indicate the lowest datum still within 1.5 interquartile range (IQR) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile. Sample sizes: n = 1,139 (Y503D), n = 24 (K476C), n = 7 (wild type) runs. d, Translocation example for ClpB(K476C). Scale bars correspond to 200 aa and 10 s. e, Translocation example for wild-type ClpB with the DnaK system (DnaK, DnaJ and GrpE). Scale bars correspond to 200 aa and 5 s. f, g, Absolute ATPase rate (f) and ATPase substrate-stimulation (g) for the three ClpB variants and different substrate conditions (mean ± s.d.). ATPase activity is higher and more strongly stimulated for Y503D, followed by K476C and wild type. The lower activities observed in the presence of denatured MBP–DM with respect to casein may reflect lower affinity and lower concentrations due to aggregation. The ATPase activity assay was repeated three times for all conditions in f and g, except for K476C, WT + MBP2 and Y503D + casein, for which it was repeated two times.
