Fig. 2.

Interaction of client proteins with the isolated NTD of ClpB. (A) Overlay of ¹H–¹⁵N HSQC spectra of ClpB NTD alone (in black) and in the presence of twofold excess of PhoA^1-121 (red), PhoA^119-243 (yellow), PhoA^244-349 (purple), PhoA^349-471 (green), α-casein (maroon), or p13 (cyan). Datasets were recorded at 11.7 T, 55 °C (500-MHz ¹H frequency). (B, Top) ClpB NTD surface representation (PDB ID code 1QVR) (10) with NTD residues found by NMR to interact with the unstructured substrates from (A) colored orange. (B, Bottom) ClpB NTD structure colored by residue hydrophobicity (53) (white to red gradient) showing that the NTD substrate binding site is enriched in hydrophobic residues. (C) ¹H–¹⁵N HSQC spectrum of an unstructured substrate protein, Sic1, alone (black) and upon interaction with ClpB NTD (red), 18 °C. Sic1 assignments (26) are indicated. (D) Percentage values of ClpB-client protein amino acids that show CSPs in the presence of NTD (shifts greater than 1 SD from the average). Values are normalized against the total number of each amino acid in the substrate sequences. From the data, NTD preferably binds to hydrophobic residues in its client proteins. Assignments of cross-peaks are as indicated in A and C.