W0156

The 2.1 Å Resolution Structure of Human Mitochondrial ATP-dependent ClpP Protease. Bijan Ahvazi ^1,2,3, Sung Gyun Kang⁴, Mark Thompson¹, Michael R. Maurizi⁴, Timothy Mueser¹, ¹Laboratory of Structural Biology Research, ²Laboratory of Skin Biology, ³Laboratory of X-ray Crystallography/Office of Science and Technology, National Inst. of Arthritis and Musculoskeletal and Skin Diseases, ⁴Laboratory of Cell Biology, National Cancer Inst., National Insts. of Health, Bethesda, MD 20892-8023, U.S.A.

The crystal structure of the human mitochondrial ClpP protease (hClpP) has been solved at a resolution of 2.1 Å. Human ClpXP is a bipartite protease responsible in mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments. The ClpX (on chromosome 15q22.1) component of the ClpXP is a hexameric ring ATPase which combines a molecular chaperone and a self-compartmentalizing protease ClpP (on chromosome 19) to create complexes that catalyze regulated degradation of specific protein substrates. E.coli ClpA and ClpX are members of the Clp/Hsp100 family of ATP-dependent molecular chaperones and can function as autonomous chaperones. ClpA and ClpX derive from a common ancestor and belong to the AAA super-family of ATPases. Even though some members of each family can be heat-induced, genetic studies indicate that chaperonins are essential in protein folding at all temperatures. When chaperonin function is deficient, newly translocated or synthesized proteins fail to reach native form and are liable to aggregate.

The ClpP component of ClpXP is a highly conserved serine peptidase with a classical Ser-His-Asp triad in the active site with broad sequence specificity. ClpP subunits are arranged in a seven-membered ring and the holoenzyme is formed by face to face stacking of two heptameric rings which enclose a central chamber containing the active sites. The active sites of ClpP are located within an aqueous cavity formed by the conjunction of the two rings. The access to the proteolytic chamber of ClpP are limited by narrow axial portals on both sites, only short unstructured peptides can be degraded by ClpP alone. This mechanism serves to protect cellular protein from cleavage unless they are recognized, denatured and translocated into ClpP chamber by an associated ATPase such as ClpX. ClpP can form proteolytic complexes with ClpA and ClpX, which bind to the exterior surface of the ClpP rings. ClpA and ClpX interact directly with proteins substrates and function in substrate discrimination. When complexed with ClpA or ClpX, ClpP can degrade large proteins, but it is necessary that the proteins be unfolded into an extended conformation to enter the ClpP active site cavity. The function of the chaperone activity of ClpA or ClpX is to unfold protein substrates and aid in their translocation of ClpP.