ClpA is an ATP-dependent chaperone that forms a hexameric ring that alone has an unfoldase activity in vitro. Two of these rings flank ClpP tetradecamer forming ClpAP protease. In this complex ClpA assists the proteolytic core by binding, unfolding and translocating substrates to the proteolytic chamber. Among proteins recognized and degraded by ClpA are those modified by C-terminal addition of the peptide sequence AANDENYALAA (SsrA tag) which is encoded by SsrA RNA. Both proteins synthesized from mRNAs lacking the stop codons and from mRNAs containing rare-codons can be tagged by the SsrA system. Thermodynamic parameters of ClpA-peptide interactions in the presence of 1mM ATPgammaS at pH 7.5 have been determined by isothermal titration calorimetry (ITC) using the 11-amino acid substrate AANDENYALAA (SsrA). Light scattering studies confirm that ClpA with 1 mM ATPgammaS maintains a hexameric form over the temperature range of 6 to 50 C. The association constant of log K = 6.70 +/- 0.6 (/M) obtained by ITC is an order of magnitude higher than that previously determined by inhibition of casein degradation. Titrations at different temperatures show that binding enthalpies decrease with increasing temperature between 4 and 28 C yielding a heat capacity change of -1.2 kcal/(K mol). The negative heat capacity change provides strong evidence for the role of hydrophobic interactions as the driving force for the association of this substrate with ClpA. In contrast to the binding enthalpy, both the affinity constant and the binding stoichiometry of 1:1 SsrA peptide : ClpA hexamer does not significantly change with temperature. In the absence of ATPgammaS, no significant binding of the SsrA peptide to ClpA could be detected by ITC. Dansylated SsrA (DNS-SsrA) has been used in fluorescence titrations to obtain the association constant by an independent method. In fluorescence titrations at 28 C, log K = 6.9 which is in agreement with the value obtained by ITC in this temperature. Furthermore, the binding stoichiometry of 1:1 DNS-SsrA peptide : ClpA hexamer was confirmed by the continuous variation (Job) plot, applying the dansyl-SsrA fluorescence. The DNS-SsrA has been found also to be competitively displaced by larger substrates of ClpA such as RepA or 3betaSsrA (MW 3200) but not by the nonbinding peptide AANDENYALDD (DD-SsrA). The binding of SsrA to a single site in the 505 kD hexamer is subject to several interpretations. Binding to a single subunit site could produce a high degree of negative cooperativity within the hexamer. Alternatively, the peptide could bind to a symmetrical site formed by all six subunits, presumably in the axial channel. The latter possibility is favored because it also explains why the hexameric structure of ClpA is required for binding SsrA. Future experiments are planned to obtain the thermodynamic parameters for binding 3betaSsrA to ClpA and to determine what the proton release or uptake is for substrate binding to ClpA.
