Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions

Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer

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Examination of the Polypeptide Substrate Specificity for <i>Escherichia coli</i> 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. <i>Escherichia coli</i> 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 <i>E. coli</i> 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

<i>Escherichia coli</i> DnaK Allosterically Modulates ClpB between High- and Low-Peptide Affinity States

ClpB and DnaKJE provide protection to <i>Escherichia coli</i> cells during extreme environmental stress. Together, this co-chaperone system can resolve protein aggregates, restoring misfolded proteins to their native form and function in solubilizing damaged proteins for removal by the cell’s proteolytic systems. DnaK is the component of the KJE system that directly interacts with ClpB. There are many hypotheses for how DnaK affects ClpB-catalyzed disaggregation, each with some experimental support. Here, we build on our recent work characterizing the molecular mechanism of ClpB-catalyzed polypeptide translocation by developing a stopped-flow FRET assay that allows us to detect ClpB’s movement on model polypeptide substrates in the absence or presence of DnaK. We find that DnaK induces ClpB to dissociate from the polypeptide substrate. We propose that DnaK acts as a peptide release factor, binding ClpB and causing the ClpB conformation to change to a low-peptide affinity state. Such a role for DnaK would allow ClpB to rebind to another portion of an aggregate and continue nonprocessive translocation to disrupt the aggregate