Our research is focused on the structure/function relationships of the ATP-dependent Clp and Lon proteases, which degrade important regulatory proteins as well as damaged and denatured proteins in E. coli and human cells. Wild-type Lon can be expressed using a Vaccinia expression system, and the protein is targeted to mitochondria and processed. Processing of human Lon appears to be autocatalytic, because a mutant Lon in which the active site serine residue has been altered is targeted to mitochondria but is not processed and co-expression of the mutant and the wild-type Lon proteases leads to processing of the mutant. Expression of wild-type Lon in HeLa cells causes the cells to round up and lose viability, whereas expression of the serine mutant has no deleterious effects. This system will be exploited to define functional changes in different Lon mutants and to identify physiological targets of human Lon protease. Stable transfectants of human Lon have been obtained using a construct with MDR1 as the dominant selectable marker. In this system also, high level expression of the inactive mutant but not of wild-type Lon has been possible. We have cloned and expressed E. coli ClpX, a member of the Clp family of ATPases. ClpX has ATP-dependent chaperone activity and is required for some specific ATP-dependent proteolytic activities dependent on ClpP. Gel filtration and electron microscopy show that ClpX subunits (Mr 46,000) associate to form a six- membered ring (Mr 280,000) that is stabilized by binding of ATP. In the presence of ATP, hexameric ClpX interacts with ClpP, a tetradecamer composed of superimposed seven-membered rings, to form a stable complex that can be isolated by gel filtration. In the complex, the rings of ClpP are flanked on each side by a single ring of ClpX. A symmetry mismatch thus exists between the seven-membered rings of ClpP and the six-membered ATPase rings for both ClpXP and ClpAP. Competition studies showed that ClpX and ClpA have nearly equal affinity for binding to ClpP, however no evidence for mixed complexes of ClpA, ClpX, and ClpP were observed suggesting that binding of a specific ATPase to one face of ClpP favors binding of a like ATPase to the opposite face of ClpP. The oligopeptide, FAPHMALVPV, is cleaved by ClpXP in the presence of non- hydrolyzable analogs of ATP with a turnover number of 10,000 min-1 (per tetradecamer of ClpP), indicating that ClpX, as does ClpA, allosterically activates affects ClpP to make the active site more accessible and to potentiate the catalytic efficiency of the proteolytic active site. Studies of subunit interactions in ClpP indicate that contacts between rings can be disrupted without breaking the subunit interactions within the heptameric rings. Treatment with high salt concentrations and low temperature lead to reversible separation of the ClpP rings, which can reassociate with complete restoration of enzymatic activity.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC005597-07
Application #
6160928
Study Section
Special Emphasis Panel (LCB)
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Lies, Mark; Maurizi, Michael R (2008) Turnover of endogenous SsrA-tagged proteins mediated by ATP-dependent proteases in Escherichia coli. J Biol Chem 283:22918-29
Rotanova, Tatyana V; Botos, Istvan; Melnikov, Edward E et al. (2006) Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci 15:1815-28
Szyk, Agnieszka; Maurizi, Michael R (2006) Crystal structure at 1.9A of E. coli ClpP with a peptide covalently bound at the active site. J Struct Biol 156:165-74
Kang, Sung Gyun; Maurizi, Michael R; Thompson, Mark et al. (2004) Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP. J Struct Biol 148:338-52
Botos, Istvan; Melnikov, Edward E; Cherry, Scott et al. (2004) Crystal structure of the AAA+ alpha domain of E. coli Lon protease at 1.9A resolution. J Struct Biol 146:113-22
Botos, Istvan; Melnikov, Edward E; Cherry, Scott et al. (2004) The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site. J Biol Chem 279:8140-8
Ishikawa, Takashi; Maurizi, Michael R; Steven, Alasdair C (2004) The N-terminal substrate-binding domain of ClpA unfoldase is highly mobile and extends axially from the distal surface of ClpAP protease. J Struct Biol 146:180-8
Xia, Di; Esser, Lothar; Singh, Satyendra K et al. (2004) Crystallographic investigation of peptide binding sites in the N-domain of the ClpA chaperone. J Struct Biol 146:166-79
Ortega, Joaquin; Lee, Hyun Sook; Maurizi, Michael R et al. (2004) ClpA and ClpX ATPases bind simultaneously to opposite ends of ClpP peptidase to form active hybrid complexes. J Struct Biol 146:217-26
Maurizi, Michael R; Xia, Di (2004) Protein binding and disruption by Clp/Hsp100 chaperones. Structure 12:175-83