Enzymatic and structural similarities between the Escherichia coli ATP- dependent proteases, ClpXP and ClpAP

Régis Grimaud (1, 2) , M. Kessel (1) , F. Beuron (1) , A.C. Steven (1) , M.R. Maurizi (1)

1 NIH - National Institutes of Health [Bethesda, MD, USA]
2 IPREM - Institut des sciences analytiques et de physico-chimie pour l'environnement et les materiaux

Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP- dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (M(r) 46,000) associate to form a six- membered ring (M(r) ~ 280,000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP, ClpP, which is composed of two seven- membered rings stacked face-to-face, interacts with the nucleotide- stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven- membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (~2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min-1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

Mots clés: adenosine triphosphatase; bacterial enzyme; binding affinity; conformational transition; electron microscopy; enzyme activity; enzyme analysis; enzyme binding; enzyme conformation; enzyme structure; enzyme substrate complex; protein degradation; Adenosine Triphosphatases; Endopeptidase Clp; Escherichia coli; Molecular Chaperones; Serine Endopeptidases; Substrate Specificity

Domaines: Biologie structurale [q-bio.BM]; Bactériologie

Régis Grimaud, M. Kessel, F. Beuron, A.C. Steven, M.R. Maurizi. Enzymatic and structural similarities between the Escherichia coli ATP- dependent proteases, ClpXP and ClpAP. Journal of Biological Chemistry, 1998, 273 (20), pp.12476-12481. ⟨10.1074/jbc.273.20.12476⟩. ⟨hal-01614793⟩