Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX

Clp ATPases are a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX is a representative member of these ATPases; it consists of two domains, a zinc-binding domain (ZBD) that forms dimers and a AAA⁺ ATP-binding domain that arranges into a hexamer. Analysis of the binding preferences of these two domains in ClpX revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA⁺ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of the ClpX dimeric cofactor, SspB₂, on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB₂ toward ZBD₂ and the geometry of the SspB₂-ZBD₂ complex were investigated by using the newly developed quantitative optical biosensor method of dual polarization interferometry. The data suggest a model for the interaction between SspB₂ and the ClpX hexamer.