ATP Binds to Proteasomal ATPases in Pairs with Distinct Functional Effects, Implying an Ordered Reaction Cycle

In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules or two ATPγS plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.

[1]  Thomas Walz,et al.  ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. , 2005, Molecular cell.

[2]  Yigong Shi,et al.  Mechanism of substrate unfolding and translocation by the regulatory particle of the proteasome from Methanocaldococcus jannaschii. , 2009, Molecular cell.

[3]  Friedrich Förster,et al.  Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution , 2010, Proceedings of the National Academy of Sciences.

[4]  H. Ploegh,et al.  Multiple associated proteins regulate proteasome structure and function. , 2002, Molecular cell.

[5]  A. Wilkinson,et al.  AAA+ superfamily ATPases: common structure–diverse function , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

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

[7]  Yigong Shi,et al.  Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii. , 2009, Molecular cell.

[8]  E. Morris,et al.  Structure of the Human 26S Proteasome , 2008, Journal of Biological Chemistry.

[9]  A. Goldberg.  Nobel Committee Tags Ubiquitin for Distinction , 2005, Neuron.

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

[11]  M. Buck,et al.  Mechanism of homotropic control to coordinate hydrolysis in a hexameric AAA+ ring ATPase. , 2008, Journal of molecular biology.

[12]  S. Lindquist,et al.  Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. Lupas,et al.  AAA proteins. , 2002, Current opinion in structural biology.

[14]  A. Horwitz,et al.  ATP-induced Structural Transitions in PAN, the Proteasome-regulatory ATPase Complex in Archaea* , 2007, Journal of Biological Chemistry.

[15]  S. Walter,et al.  Substrate Binding to the Molecular Chaperone Hsp104 and Its Regulation by Nucleotides* , 2005, Journal of Biological Chemistry.

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

[17]  J. E. D. Esteves da Silva,et al.  Pyrophosphate and tripolyphosphate affect firefly luciferase luminescence because they act as substrates and not as allosteric effectors , 2008, The FEBS journal.

[18]  S. Gygi,et al.  Isolation of mammalian 26S proteasomes and p97/VCP complexes using the ubiquitin-like domain from HHR23B reveals novel proteasome-associated proteins. , 2009, Biochemistry.

[19]  M. Glickman,et al.  Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites , 2007, Nature Structural &Molecular Biology.

[20]  A. Goldberg,et al.  Proteasomes and their associated ATPases: a destructive combination. , 2006, Journal of structural biology.

[21]  Hongtao Yu,et al.  ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. , 2006, Molecular cell.

[22]  B. Ames.  ASSAY OF INORGANIC PHOSPHATE, TOTAL PHOSPHATE AND PHOSPHATASE , 1966 .

[23]  P. Freemont,et al.  Analysis of Nucleotide Binding to P97 Reveals the Properties of a Tandem AAA Hexameric ATPase , 2008, Journal of Biological Chemistry.

[24]  Antonio Sillero,et al.  Dehydroluciferyl‐AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase , 1998, FEBS letters.

[25]  A. Goldberg,et al.  ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. , 2003, Molecular cell.

[26]  A. Ciechanover,et al.  The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. , 2002, Physiological reviews.

[27]  Robert T Sauer,et al.  Asymmetric nucleotide transactions of the HslUV protease. , 2008, Journal of molecular biology.

[28]  T. Langer,et al.  An intersubunit signaling network coordinates ATP hydrolysis by m-AAA proteases. , 2009, Molecular cell.

[29]  M. Habeck,et al.  Structure and activity of the N-terminal substrate recognition domains in proteasomal ATPases. , 2009, Molecular cell.

[30]  Soyeon Park,et al.  Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry. , 2007, Molecular cell.

[31]  A. Goldberg,et al.  PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone , 2000, Nature Cell Biology.

[32]  S. Gygi,et al.  Hexameric assembly of the proteasomal ATPases is templated through their C-termini , 2009, Nature.

[33]  Ronald T. Raines,et al.  Fluorescence polarization assay to quantify protein-protein interactions. , 2004, Methods in molecular biology.

[34]  Yifan Cheng,et al.  Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. , 2008, Molecular cell.

[35]  A. Goldberg,et al.  Binding of nucleotides to the ATP-dependent protease La from Escherichia coli. , 1987, The Journal of biological chemistry.

[36]  S. Glynn,et al.  Structures of Asymmetric ClpX Hexamers Reveal Nucleotide-Dependent Motions in a AAA+ Protein-Unfolding Machine , 2009, Cell.

[37]  Yifan Cheng,et al.  Interactions of PAN's C‐termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions , 2010, The EMBO journal.

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

[39]  Friedrich Förster,et al.  Insights into the molecular architecture of the 26S proteasome , 2009, Proceedings of the National Academy of Sciences.

[40]  Jimin Wang,et al.  Heterohexameric ring arrangement of the eukaryotic proteasomal ATPases: implications for proteasome structure and assembly. , 2010, Molecular cell.

[41]  Tania A. Baker,et al.  Asymmetric Interactions of ATP with the AAA+ ClpX6 Unfoldase: Allosteric Control of a Protein Machine , 2005, Cell.

[42]  R. Huber,et al.  Structure of 20S proteasome from yeast at 2.4Å resolution , 1997, Nature.

[43]  W. Baumeister,et al.  26S proteasome structure revealed by three-dimensional electron microscopy. , 1998, Journal of structural biology.

[44]  R. Huber,et al.  Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. , 1995, Science.

[45]  Tania A. Baker,et al.  Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines , 2005, Nature.