Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine.
ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.
Burton
R E
RE
Department of Biology, Massachusetts Institute of Technology and Howard Hughes Medical Institute, Cambridge, MA 02139, USA.
Siddiqui
S M
SM
Kim
Y I
YI
Baker
T A
TA
Sauer
R T
RT
D000251:Adenosine Triphosphatases / Q000378:metabolism* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D049071:Endopeptidase Clp; D017510:Protein Folding; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism* / Q000502:physiology; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C096222:tmRNA; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases