Fig. 5.
Substrate binding to hexameric ClpB. (A and B) Chemical shift perturbations induced by α-casein binding to (A) [2H, 13CH3-ILVM]-labeled isolated NTD and (B) segmentally [2H, 13CH3-ILVM]-ClpBNTD,[2H, 12C]-ClpBΔN labeled ClpBY243A. CSPs are defined by the relation , where ΔδH and ΔδC are methyl 1H and 13C chemical-shift changes between apo and bound forms of the protein and α (β) is 1 SD of the methyl 1H (13C) chemical shifts deposited in the Biological Magnetic Resonance Data Bank [α is 0.29 (I), 0.28 (L), 0.27 (V), and 0.41 (M), whereas β is 1.65 (I), 1.6 (L), 1.4 (V), and 1.54 (M)]. (C and D) Titration curves for NTD (C) and segmentally labeled ClpBY243A (D), 55 °C. Either 1H or 13C chemical-shift changes, Δδ (in parts per million), are plotted as a function of α-casein concentration, from which KD values for the NTD–casein (C) and ClpBY243A–casein (D) interactions were obtained as described in the text. (E) CSPs for segmentally ILVM-labeled ClpBWT at the endpoint of the titration with α-casein, 55 °C. Residues in the NTD-NBD1 linker showing measurable CSPs for ClpBWT, but not for ClpBY243A, are colored in green. Inset shows cartoon representation highlighting the NTD and NBD1 domains of ClpB, with stick representations of NTD substrate-binding methyl residues (blue) and linker methyl residues (green). (F and G) Titration of α-casein into (F) ATP-bound and (G) ADP-bound hexameric ClpB with segmentally methyl-labeled NTD. Δδ as a function of α-casein concentration for residues L132, L135, and V141 (reporting on binding to the tyrosine loops) are shown in green. The corresponding curves for binding of α-casein to the NTDs of the hexamer are shown in blue. All fits (solid lines) in F and G correspond to the case of three α-casein molecules bound per hexamer that produced the best fits (Fig. S6). Calculated dissociation constants for the α-casein interaction with hexameric ClpB are reported in Table S2.