Functional Domains of the ClpA and ClpX Molecular Chaperones Identified by Limited Proteolysis and Deletion Analysis

• Published in: The Journal of biological chemistry 2001-08, Vol.276 (31), p.29420-29429
• Main Authors: Singh, S K, Rozycki, J, Ortega, J, Ishikawa, T, Lo, J, Steven, A C, Maurizi, M R
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Adenosine Triphosphatases - ultrastructure; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Amino Acid Sequence; ATPases Associated with Diverse Cellular Activities; Base Sequence; Binding Sites; ClpA protein; ClpX protein; Conserved Sequence; DNA Primers; Endopeptidase Clp; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Kinetics; Macromolecular Substances; Microscopy, Electron; Molecular Chaperones - chemistry; Molecular Chaperones - metabolism; Molecular Sequence Data; Mutagenesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - ultrastructure; Sequence Deletion; Serine Endopeptidases - chemistry; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Serine Endopeptidases - ultrastructure; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M103489200

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATPγS binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an α-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXΔN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.