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.

[1]  M. Cox,et al.  RuvB protein-mediated ATP hydrolysis: functional asymmetry in the RuvB hexamer. , 1995, Biochemistry.

[2]  Michael R Sawaya,et al.  Crystal Structure of T7 Gene 4 Ring Helicase Indicates a Mechanism for Sequential Hydrolysis of Nucleotides , 2000, Cell.

[3]  M. Hingorani,et al.  Nucleotide binding studies of bacteriophage T7 DNA helicase-primase protein. , 1995, Biophysical journal.

[4]  J. Wang,et al.  Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism. , 2001, Structure.

[5]  Robert T Sauer,et al.  Bivalent tethering of SspB to ClpXP is required for efficient substrate delivery: a protein-design study. , 2004, Molecular cell.

[6]  R. Sauer,et al.  The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. , 1998, Genes & development.

[7]  Greg L. Hersch,et al.  Communication between ClpX and ClpP during substrate processing and degradation , 2004, Nature Structural &Molecular Biology.

[8]  Dong-Eun Kim,et al.  Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA* , 2002, The Journal of Biological Chemistry.

[9]  W. Wooster,et al.  Crystal structure of , 2005 .

[10]  J. Kuriyan,et al.  Mechanism of Processivity Clamp Opening by the Delta Subunit Wrench of the Clamp Loader Complex of E. coli DNA Polymerase III , 2001, Cell.

[11]  Amnon Horovitz.  Faculty Opinions recommendation of Communication between ClpX and ClpP during substrate processing and degradation. , 2004 .

[12]  P. Boyer.  The ATP synthase--a splendid molecular machine. , 1997, Annual review of biochemistry.

[13]  Kazuo Sutoh,et al.  Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. , 2004, Biochemistry.

[14]  T. Baker,et al.  Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA+ ClpXP protease. , 2004, Molecular cell.

[15]  J. Wang,et al.  Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU. , 2001, Structure.

[16]  L. Esser,et al.  Crystal Structure of ClpA, an Hsp100 Chaperone and Regulator of ClpAP Protease* , 2002, The Journal of Biological Chemistry.

[17]  Dong Young Kim,et al.  Crystal Structure of ClpX Molecular Chaperone from Helicobacter pylori* , 2003, Journal of Biological Chemistry.

[18]  T. Baker,et al.  Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation. , 2004, Journal of structural biology.

[19]  H. Shinagawa,et al.  Direct evidence that a conserved arginine in RuvB AAA+ ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Wah Chiu,et al.  The Structure of ClpB A Molecular Chaperone that Rescues Proteins from an Aggregated State , 2003, Cell.

[21]  John F Hunt,et al.  ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. , 2002, Molecular cell.

[22]  B. Stitt.  Escherichia coli transcription termination protein rho has three hydrolytic sites for ATP. , 1988, The Journal of biological chemistry.

[23]  S. Bell,et al.  Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication. , 2001, Molecular cell.

[24]  T. Baker,et al.  Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Robert T Sauer,et al.  Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  G. Piszczek,et al.  The Molecular Chaperone, ClpA, Has a Single High Affinity Peptide Binding Site per Hexamer* , 2005, Journal of Biological Chemistry.

[27]  Robert Huber,et al.  The structures of HslU and the ATP-dependent protease HslU–HslV , 2000, Nature.

[28]  T. Baker,et al.  Characterization of a specificity factor for an AAA+ ATPase: assembly of SspB dimers with ssrA-tagged proteins and the ClpX hexamer. , 2002, Chemistry & biology.

[29]  T. Baker,et al.  Dynamics of substrate denaturation and translocation by the ClpXP degradation machine. , 2000, Molecular cell.

[30]  J. Tainer,et al.  Structure and mechanism of the RuvB Holliday junction branch migration motor. , 2001, Journal of molecular biology.

[31]  Greg L. Hersch,et al.  Sculpting the Proteome with AAA+ Proteases and Disassembly Machines , 2004, Cell.

[32]  T. Baker,et al.  Role of the processing pore of the ClpX AAA+ ATPase in the recognition and engagement of specific protein substrates. , 2004, Genes & development.

[33]  D. Gai,et al.  Mechanisms of Conformational Change for a Replicative Hexameric Helicase of SV40 Large Tumor Antigen , 2004, Cell.

[34]  J. Tkach,et al.  Crowbars and ratchets: hsp100 chaperones as tools in reversing protein aggregation. , 2001, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[35]  M. Cox,et al.  Allosteric effects of RuvA protein, ATP, and DNA on RuvB protein-mediated ATP hydrolysis. , 1996, Biochemistry.

[36]  T. Baker,et al.  Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine , 2001, The EMBO journal.

[37]  R. Huber,et al.  The structures of HsIU and the ATP-dependent protease HsIU-HsIV. , 2000, Nature.

[38]  T. Baker,et al.  Energy‐dependent degradation: Linkage between ClpX‐catalyzed nucleotide hydrolysis and protein‐substrate processing , 2003, Protein science : a publication of the Protein Society.

[39]  Robert T Sauer,et al.  Nucleotide-dependent substrate recognition by the AAA+ HslUV protease , 2005, Nature Structural &Molecular Biology.

[40]  A. Steven,et al.  Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP. , 2000, Molecular cell.

[41]  Christine B. Trame,et al.  Crystal and Solution Structures of an HslUV Protease–Chaperone Complex , 2000, Cell.

[42]  Jimin Wang,et al.  The Structure of ClpP at 2.3 Å Resolution Suggests a Model for ATP-Dependent Proteolysis , 1997, Cell.

[43]  A. di Pietro,et al.  The Conserved Glutamate Residue Adjacent to the Walker-B Motif Is the Catalytic Base for ATP Hydrolysis in the ATP-binding Cassette Transporter BmrA* , 2003, Journal of Biological Chemistry.

[44]  V. Shoshan-Barmatz,et al.  ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase. , 2003, The Biochemical journal.