Mechanism and consequences of Mu transpososome remodeling by the ClpX chaperone

Abstract

E. coli ClpX is a member of the Clp/Hsp100 family of ATPases that remodel multi-component complexes and facilitate ATP-dependent protein degradation. Protein remodelers alter the biological activity of their substrates, typically by changing the quaternary structure of their target proteins. ClpX remodels protein-DNA complexes, termed transpososomes, made during recombination of the phage Mu. When recombination is complete, the core four-subunit transpososome complex does not spontaneously release the DNA; transposase remains so stably bound that subsequent replication of the Mu genome is inhibited. To understand how ClpX releases the replication block without destroying the transpososomes, we characterized the mechanism and products of transpososome remodeling. To better understand the mechanism ClpX uses to facilitate remodeling, I first participated in a collaborative project that defined major biochemical reaction steps involved in protein degradation by ClpX and its associated peptidase ClpP. These experiments demonstrated that ClpX acts as a powerful unfolding enzyme during degradation reactions. Then, I asked whether this same unfolding activity was also responsible for transpososome remodeling. These experiments demonstrated that ClpX can unfold transposase monomers, and that it exerts a conformational change in transposase subunits during remodeling. However, only subunits directly contacted by ClpX undergo a conformational change. Analysis of the minimal interaction necessary between ClpX and the complex revealed that recognition of one subunit in the complex is sufficient for remodeling.

(cont.) These results establish that the unfolding activity of ClpX is sufficient to explain its role in both complex destabilization and proteolysis. To understand how ClpX could use such a destructive activity for a non-destructive process, I characterized the products of the remodeling reaction by following the fate of both the protein subunits and the Mu DNA. Whereas some subunits remain associated with the recombined DNA, release of a subset of subunits accompanies remodeling. The products of remodeling were unchanged even when a degradation-specific signal was placed on the transposase or when another unfolding enzyme was used for the reaction. Thus, we propose that physical properties of the target complex direct the configuration of the remodeled products. This work provides a model for how unfolding enzymes can alter biological complexes in a non-destructive manner.