Design framework of the MuA remodeling signal that confers preferential complex disassembly by the AAA+ unfoldase ClpX

Abstract

The cell employs many classes of molecular chaperones to facilitate proteins in adopting the proper structure and preventing non-functional and potentially toxic non-native states. The Clp/Hsp100 family of ATPases are unfolding chaperones that remodel macromolecular complexes and facilitate ATP-dependent protein degradation. They are members of the superfamily of AAA+ enzymes (ATPases Associated with various cellular Activities), which is conserved across all kingdoms of life. Efficient selection of multimeric protein complexes over constituent subunits is key to successful remodeling and disassembly reactions. Using E.coli ClpX as a model for AAA+ ATPases, I characterized the mechanism by which ClpX discriminates between two oligomeric states of one of its natural multimeric substrates, phage MuA tranposase. I elucidated many strategies for ClpX's preference for the assembled Mu transpososome (MuA complex) over unassembled subunits. First, the target substrate makes multiple weak interactions with the AAA+ ATPase via the pore in the conserved ATPase domain and a class-specific auxiliary domain. Second, recognition tags should be at the weaker end of the affinity spectrum to allow effective synergy of multiple tags in the assembled complex. Third, multimeric complexes can "divide the labor" of making these interactions among their subunits. Thus the holistic complex-specific targeting signal is accessible only in the assembled complex. The work of this thesis has provided a framework to understand the design of recognition signals that specify and target macromolecular complexes to unfolding chaperones and remodelers of the AAA+ superfamily.