Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine.
ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02319, USA.
Greg L
GL
Hersch
Randall E
RE
Burton
Daniel N
DN
Bolon
Tania A
TA
Baker
Robert T
RT
Sauer
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation / Q000502:physiology; D000495:Allosteric Site / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism*; D025243:Bacterial Proton-Translocating ATPases / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism*; D004734:Energy Metabolism / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009711:Nucleotides; D021122:Protein Subunits; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D025243:Bacterial Proton-Translocating ATPases