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C-terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding
Shilpa A Joshi1, Tania A Baker, Robert T Sauer
1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
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.
Insights
ClpX protein mutations impair its ability to unfold and disassemble protein complexes. These ClpX mutants show defective ATP hydrolysis, impacting cellular protein homeostasis.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Enzymology
Background:
- ClpX is an AAA+ ATPase crucial for protein denaturation and complex disassembly.
- It possesses an alphabeta ATPase domain and an alpha-helical C-terminal domain.
- Understanding ClpX function is vital for cellular protein quality control.
Purpose of the Study:
- To investigate the role of ClpX's C-terminal domain in its enzymatic activity.
- To identify specific mutations affecting ClpX's protein disassembly and unfolding functions.
- To elucidate the mechanism of ATP hydrolysis regulation by substrate binding.
Main Methods:
- Site-directed mutagenesis of the ClpX C-terminal domain.
- In vivo screening for defective disassembly phenotypes.
- In vitro characterization of purified mutant ClpX enzymes, including ATPase assays and substrate binding studies.
Main Results:
- Seven mutant ClpX proteins with defective phenotypes were identified.
- Three mutants (L381K, D382K, Y385A) exhibited reduced in vitro disassembly and unfolding activity.
- Substrate binding inhibited ATP hydrolysis in these mutants, unlike wild-type ClpX.
- Mutations affected inter-subunit communication within the ClpX hexamer.
Conclusions:
- Specific residues in the ClpX C-terminal domain are essential for substrate engagement and ATP hydrolysis.
- These mutations disrupt the communication between nucleotide-binding sites in the ClpX hexamer.
- The findings provide insights into the mechanism of AAA+ ATPase function and regulation by ClpX.