C‐terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding

Abstract

SummaryClpX mediates ATP-dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + + + + family members, ClpX contains an a a a ab b b b ATPase domain and an a a a a -helical Cterminal 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.

Crystal Structure of ClpX Molecular Chaperone from Helicobacter pylori

“…This is also true for HslU and ClpA D2, but not for ClpA D1 (17, 18, 30). A recent experiment also supports that substrate binding to the SSD domain could modulate the ATP hydrolysis and unfolding activity by demonstrating that the mutations in the SSD domain uncouple the substrate binding from ATP hydrolysis required for unfolding (38). The crystal structure of Hp ClpX-ASD also reveals that the sensor I (␤6; Fig.…”

Stepwise Activity of ClpY (HslU) Mutants in the Processive Degradation of Escherichia coli ClpYQ (HslUV) Protease Substrates

“…ClpY(Y408A), with a mutation in domain C, associated with SulA without efficiently degrading it. A ClpX mutant with a single amino acid substitution in its domain C at position 385 (tyrosine-to-alanine substitution) binds to its substrate but lacks unfolding activity (13). Yet the real mechanism behind the observation is not clear.…”

“…Moreover, ClpY (T87I), with a mutation in the ATP-binding site, did not undergo a conformational change to its hexameric form for association with ClpQ. Tyrosine residue 408 in ClpY, like residue 385 in ClpX (13), was necessary for self-oligomerization, and this activity is likely important for in vivo protein-subunit stability.…”

TorsinA and the Pathophysiology of DYT1 Dystonia

“…The AAA+ module contains four conserved motifs: a Walker A motif that regulates nucleotide binding; a Walker B motif that is crucial for ATPase activity; a sensor 1 that is critical for nucleotide hydrolysis; and sensor 2 that is essential for nucleotide binding and hydrolysis (Ozelius et al, 1997;Neuwald et al, 1999;Ozelius et al, 1999;Joshi et al, 2003;Kamm et al, 2004;Hanson and Whiteheart, 2005;Callan et al, 2007). Unlike the other three motifs, which are very conserved and play similar functions between AAA+ proteins, sensor 2 determines the specificity of the protein (Joshi et al, 2003). Sensor 2 is also a very important domain to regulate subunit and/or substrate conformational changes.…”

“…Sensor 2 is also a very important domain to regulate subunit and/or substrate conformational changes. It's a function-determined domain for AAA+ proteins because AAA+ proteins assemble into hexamers to function, and the most essential function is to regulate conformational changes of the substrates (Neuwald et al, 1999;Joshi et al, 2003;Hanson and Whiteheart, 2005). Members of the AAA+ family function as molecular chaperones for protein quality control (protein complex assembly, operation, disassembly, protein folding, unfolding, and degradation), membrane fusion and vesicular transport, and cytoskeletal regulation (Neuwald et al, 1999;Vale, 2000;Ogura and Wilkinson, 2001), all of these activities might provide insight regarding the function of torsinA.…”