Our research is focused on the mechanisms of selective protein degradation and the structure/function relationships of the ATP-dependent Lon and Clp proteases. Lon and Clp are found in all organisms, where they help regulate the levels of important proteins and contribute to protein quality control pathways. These complex proteases are assemblies of multi-domain components with at least two types of activity. One component binds specific motifs in proteins and has molecular chaperone and protein unfoldase activity. The other component is a protease with a sequestered active site that is accessible through narrow channels that permit passage of proteins only in an extended conformation. Electron microscopy of ClpAP and ClpXP has provided a structural model for these and other ATP-dependent proteases. ClpA is a hexamer with two chaperone domains. It associates with ClpP, a double-layered heptameric ring with proteolytic active sites located in an internal chamber between the rings. ClpA also has an internal chamber where proteins may be unfolded or sequestered prior to transfer to ClpP. We have made substantial progress in structure determination of ClpA. Working with Dr. Di Xia, a PI in the Laboratory of Cell Biology, we have determined a high resolution crystal structure for ClpA and the N-terminal domain of ClpA. Both domains of ClpA have folds that place them in the AAA super-family of proteins, a diverse group of proteins with important unfolding and disassembly activity in all living cells. The structure has provided details of the positions and interactions of important functional motifs in ClpA and has improved our understanding of the domain organization, domains interactions, and conformational changes that are important for its catalytic activity. The two chambers of within ClpA have surface properties that are largely hydrophobic, but with bands of positive and negative charges at different latitudes along the six-fold axis. The N-terminal domain of ClpA has a novel fold that may enable it to interact with substrates or adaptor proteins that mediate its access to protein substrates. We have found that one such adaptor, ClpS, which others reported could modify substrate selection by ClpA, acts on ClpA only when a functional N-domain is present. Biochemical studies reveal a direct interaction between ClpS and the ClpA N-domain. ClpA apparently functions in different regulatory pathways depending on competition between substrates or adaptor proteins. Electron microscopy of complexes with fusion protein that are partially translocated and degraded have shown that substrates migrate from a binding site on the apical surface of the ATPase to a position over an axial channel, and thereafter are transferred to the interior of the complex. Protein bound on one side of the complex can be translocated into ClpP while another substrate remains bound on the chaperone at the other end. The ClpA crystal structure reveals a number of contacts between the two ATP domains which would enable communication between the domains and provide a mechanism for reciprocal translocation of substrates from either end of the complex. Human ClpP and human ClpX have been expressed and purified. The crystal structure of hClpP is virtually identical to that of E. coliClpP. hClpP has a C-terminal extension which occupies a position on the lateral surface of the double-layered ring. This extension has a large effect on the hydrodynamic properties of hClpP and affects its basal peptidase activity. hClpX activates protein degradation by hClpP, the first time that enzymatic activity has been demonstrated for the mammalian ClpXP complex. The ability to sequester substrates within the ClpP chamber is being exploited to identify in vivotargets of both the human and bacterial Clp proteases.
