Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX.
The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, including CLPXP, some of which contribute to protein maturation and quality control. Currently however, the substrates and the physiological roles of mitochondrial CLPXP in humans, has remained elusive. Similarly, the mechanism by which these ATP-dependent proteases recognize their substrates currently remains unclear. Here we report the characterization of a Walker B mutation in human CLPX, in which the highly conserved glutamate was replaced with alanine. This mutant protein exhibits improved interaction with the model unfolded substrate casein and several putative physiological substrates in vitro. Although this mutant lacks ATPase activity, it retains the ability to mediate casein degradation by hCLPP, in a fashion similar to the small molecule ClpP-activator, ADEP. Our functional dissection of hCLPX structure, also identified that most model substrates are recognized by the N-terminal domain, although some substrates bypass this step and dock, directly to the pore-1 motif. Collectively these data reveal, that despite the difference between bacterial and human CLPXP complexes, human CLPXP exhibits a similar mode of substrate recognition and is deregulated by ADEPs.
Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
Bradley R
BR
Lowth
Janine
J
Kirstein-Miles
Tamanna
T
Saiyed
Heike
H
Brötz-Oesterhelt
Richard I
RI
Morimoto
Kaye N
KN
Truscott
David A
DA
Dougan
D000818:Animals; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D013379:Substrate Specificity
C568646:ClpP protein, human; D049071:Endopeptidase Clp; C528384:CLPX protein, human