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C‐terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding
Author(s) -
Joshi Shilpa A.,
Baker Tania A.,
Sauer Robert T.
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03424.x
Subject(s) - random hexamer , mutant , biology , atp hydrolysis , protein subunit , cyclic nucleotide binding domain , atpase , biochemistry , microbiology and biotechnology , plasma protein binding , biophysics , substrate (aquarium) , aaa proteins , nucleotide , enzyme , gene , ecology
Summary ClpX mediates ATP‐dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + family members, ClpX contains an αβ ATPase domain and an α‐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.