Examination of the polypeptide substrate specificity for Escherichia coli ClpA.

Enzyme-catalyzed protein unfolding is essential for a large array of biological functions, including microtubule severing, membrane fusion, morphogenesis and trafficking of endosomes, protein disaggregation, and ATP-dependent proteolysis. These enzymes are all members of the ATPases associated with various cellular activity (AAA+) superfamily of proteins. Escherichia coli ClpA is a hexameric ring ATPase responsible for enzyme-catalyzed protein unfolding and translocation of a polypeptide chain into the central cavity of the tetradecameric E. coli ClpP serine protease for proteolytic degradation. Further, ClpA also uses its protein unfolding activity to catalyze protein remodeling reactions in the absence of ClpP. ClpA recognizes and binds a variety of protein tags displayed on proteins targeted for degradation. In addition, ClpA binds unstructured or poorly structured proteins containing no specific tag sequence. Despite this, a quantitative description of the relative binding affinities for these different substrates is not available. Here we show that ClpA binds to the 11-amino acid SsrA tag with an affinity of 200 ± 30 nM. However, when the SsrA sequence is incorporated at the carboxy terminus of a 30-50-amino acid substrate exhibiting little secondary structure, the affinity constant decreases to 3-5 nM. These results indicate that additional contacts beyond the SsrA sequence are required for maximal binding affinity. Moreover, ClpA binds to various lengths of the intrinsically unstructured protein, α-casein, with an affinity of ∼30 nM. Thus, ClpA does exhibit modest specificity for SsrA when incorporated into an unstructured protein. Moreover, incorporating these results with the known structural information suggests that SsrA makes direct contact with the domain 2 loop in the axial channel and additional substrate length is required for additional contacts within domain 1.