C-terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding.
ClpX mediates ATP-dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + family members, ClpX contains an alphabeta ATPase domain and an alpha-helical C-terminal domain. ClpX proteins with mutations in the C-terminal domain were constructed and screened for disassembly activity in vivo. Seven mutant enzymes with defective phenotypes were purified and characterized. Three of these proteins (L381K, D382K and Y385A) had low activity in disassembly or unfolding assays in vitro. In contrast to wild-type ClpX, substrate binding to these mutants inhibited ATP hydrolysis instead of increasing it. These mutants appear to be defective in a reaction step that engages bound substrate proteins and is required both for enhancement of ATP hydrolysis and for unfolding/disassembly. Some of these side chains form part of the interface between the C-terminal domain of one ClpX subunit and the ATPase domain of an adjacent subunit in the hexamer and appear to be required for communication between adjacent nucleotide binding sites.
Joshi
Shilpa A
SA
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Baker
Tania A
TA
Sauer
Robert T
RT
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities