ClpA from E. coli is an ATP-dependent chaperone that plays an important role in the cellular mechanism promoting proper protein folding, preventing aggregation, and, when present in complexes with the ClpP peptidase, ClpA functions to translocate proteins to the ClpP core to eliminate irreversibly damaged proteins. In the presence of ATP, ClpA forms a hexameric ring that alone works as an unfoldase in vitro. In E. coli, two of these rings cap the ends of ClpP protease, flanking the ends of the central unit composed of two stacked heptameric ClpP rings. In this large complex, ClpA assists the proteolytic core by binding, unfolding, and translocating proteins to the proteolytic chamber of ClpP. As a first link in this chain of events, binding properties of ClpA play a key role in the recognition of specific proteins by ClpAP complex. Binding of specific peptides sequences to ClpA also is the limiting step in the whole process of protein degradation by the ClpAP protease. It has been found that among proteins recognized and degraded by ClpA are those modified by C-terminal addition of the peptide sequence ANDENYALAA (Ssra 11aa tag) that 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. We have applied isothermal titration calorimetry (ITC) to study the interaction of ClpA and synthetic 11aa peptide corresponding to the Ssra sequence. An association constant (log K = 7.7, expressed per M peptide) has been measured by ITC, and this preliminary value is more than an order of magnitude higher than that previously estimated from kinetic measurements of the inhibition of ClpAP casein degradation by the Ssra peptide. Previous data suggested that addition of a-helical scaffold to the Ssra tag improved the binding properties of the modified peptide. We are planning to verify these results by the ITC method. Preliminary ITC measurements indicated that in the presence of ATPgammaS, the stoichiometry of binding is two peptides per ClpA hexamer. This has interesting implications in a substrate-induced asymmetry of the ClpA subunits within a hexagonal ring. Experiments to be performed in the absence of the ATPgammaS (when ClpA is in a monomer-dimer equilibrium) will give more information about the nature of peptide binding. Also, ClpA does not bind the Ssra 11aa tag peptide when its last two alanine residues are substituted by aspartic acids, and this peptide can serve as a control and reference substrate in ITC measurements.
