ATP-induced Structural Transitions in PAN, the Proteasome-regulatory ATPase Complex in Archaea

ATP binding to the PAN-ATPase complex in Archaea or the homologous 19 S protease-regulatory complex in eukaryotes induces association with the 20 S proteasome and opening of its substrate entry channel, whereas ATP hydrolysis allows unfolding of globular substrates. To clarify the conformational changes associated with ATP binding and hydrolysis, we used protease sensitivity to monitor the conformations of the PAN ATPase from Methanococcus jannischii. Exhaustive trypsin treatment of PAN generated five distinct fragments, two of which differed when a nucleotide (either ATP, ATPγS, or ADP) was bound. Surprisingly, the nucleotide concentrations altering protease sensitivity were much lower (Ka 20-40 μm) than are required for ATP-dependent protein breakdown by the PAN-20S proteasome complex (Km ∼ 300-500 μm). Unlike trypsin, proteinase K yielded several fragments that differed in the ATPγS and ADP-bound forms, and thus revealed conformational transitions associated with ATP hydrolysis. Mapping the fragments generated by each revealed that nucleotide binding and hydrolysis induce local conformational changes, affecting the Walker A and B nucleotide-binding motif, as well as global changes extending to its carboxyl terminus. The location and overlap of the fragments also suggest that the conformation of the six subunits is not identical, probably because they do not all bind ATP simultaneously. Partial nucleotide occupancy was supported by direct assays, which demonstrated that, at saturating conditions, only four nucleotides are bound to hexameric PAN. Using the protease protection maps, we modeled the conformational changes associated with ATP binding and hydrolysis in PAN based on the x-ray structures of the homologous AAA ATPase, HslU. ATP binding to the PAN-ATPase complex in Archaea or the homologous 19 S protease-regulatory complex in eukaryotes induces association with the 20 S proteasome and opening of its substrate entry channel, whereas ATP hydrolysis allows unfolding of globular substrates. To clarify the conformational changes associated with ATP binding and hydrolysis, we used protease sensitivity to monitor the conformations of the PAN ATPase from Methanococcus jannischii. Exhaustive trypsin treatment of PAN generated five distinct fragments, two of which differed when a nucleotide (either ATP, ATPγS, or ADP) was bound. Surprisingly, the nucleotide concentrations altering protease sensitivity were much lower (Ka 20-40 μm) than are required for ATP-dependent protein breakdown by the PAN-20S proteasome complex (Km ∼ 300-500 μm). Unlike trypsin, proteinase K yielded several fragments that differed in the ATPγS and ADP-bound forms, and thus revealed conformational transitions associated with ATP hydrolysis. Mapping the fragments generated by each revealed that nucleotide binding and hydrolysis induce local conformational changes, affecting the Walker A and B nucleotide-binding motif, as well as global changes extending to its carboxyl terminus. The location and overlap of the fragments also suggest that the conformation of the six subunits is not identical, probably because they do not all bind ATP simultaneously. Partial nucleotide occupancy was supported by direct assays, which demonstrated that, at saturating conditions, only four nucleotides are bound to hexameric PAN. Using the protease protection maps, we modeled the conformational changes associated with ATP binding and hydrolysis in PAN based on the x-ray structures of the homologous AAA ATPase, HslU. In both prokaryotic and eukaryotic cells, most proteins are degraded in an ATP-dependent manner (1Ciechanover A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 79-87Crossref PubMed Scopus (791) Google Scholar, 2Benaroudj N. Smith D. Goldberg A.L. The Ubiquitin Proteasome System. 2006; (, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany)Google Scholar). In eukaryotes, ATP-dependent degradation is catalyzed by the 26 S proteasome, which hydrolyzes ubiquitin-conjugated and some non-ubiquitinated proteins (3Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2215) Google Scholar, 4Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1574) Google Scholar). In this process, polyubiquitinated proteins bind the 19 S regulatory particle, which is believed to promote their unfolding and translocation into the 20 S core particle (5Kohler A. Cascio P. Leggett D.S. Woo K.M. Goldberg A.L. Finley D. Mol. Cell. 2001; 7: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), where hydrolysis to small peptides occurs. The 19 S complex contains six homologous ATPases in its base, which lies adjacent to the 20 S particle. These ATPases have been implicated in substrate recognition, unfolding, translocation, and gate opening in the outer ring of the 20 S particle (5Kohler A. Cascio P. Leggett D.S. Woo K.M. Goldberg A.L. Finley D. Mol. Cell. 2001; 7: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 7Benaroudj N. Zwickl P. Seemuller E. Baumeister W. Goldberg A.L. Mol. Cell. 2003; 11: 69-78Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Lam Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. Nature. 2002; 416: 763-767Crossref PubMed Scopus (365) Google Scholar, 9Navon A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 10Groll M. Bajorek M. Kohler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (641) Google Scholar). Although prokaryotes lack ubiquitin and 26 S proteasomes, archaebacteria do have 20 S proteasomes, whose architecture and proteolytic mechanism closely resemble those of the eukaryotic 20 S (11Zwickl P. Ng D. Woo K.M. Klenk H.P. Goldberg A.L. J. Biol. Chem. 1999; 274: 26008-26014Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Because of its greater simplicity, the archaeal 20 S proteasome has proven very useful for structural and mechanistic studies (12Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1363) Google Scholar, 13Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 14Kisselev A.F. Akopian T.N. Goldberg A.L. J. Biol. Chem. 1998; 273: 1982-1989Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The Archaea proteasome functions in protein breakdown in association with an ATPase complex, termed PAN 3The abbreviations used are: PAN, proteasome-activating nucleotidase; ATPγS, adenosine 5′-O-(thiotriphosphate); YFP, yellow fluorescent protein; AMP-PNP, adenosine 5′-(β,γ-imino) triphosphate. 3The abbreviations used are: PAN, proteasome-activating nucleotidase; ATPγS, adenosine 5′-O-(thiotriphosphate); YFP, yellow fluorescent protein; AMP-PNP, adenosine 5′-(β,γ-imino) triphosphate. (proteasome-activating nucleotidase). When mixed with the archaeal 20 S proteasome and ATP, PAN stimulates the degradation of natively unfolded proteins and peptides longer than 7 residues, whereas shorter peptides diffuse into the 20 S proteasome unassisted (11Zwickl P. Ng D. Woo K.M. Klenk H.P. Goldberg A.L. J. Biol. Chem. 1999; 274: 26008-26014Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). PAN is a hexameric ring with a central pore, and thus its general structure resembles that of the six ATPases of the 19 S complex (6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Like them, PAN exhibits several typical chaperone properties, including the ability to bind unfolded polypeptides selectively and to prevent their aggregation (15Strickland E. Hakala K. Thomas P.J. DeMartino G.N. J. Biol. Chem. 2000; 275: 5565-5572Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 16Braun B.C. Glickman M. Kraft R. Dahlmann B. Kloetzel P.M. Finley D. Schmidt M. Nat. Cell Biol. 1999; 1: 221-226Crossref PubMed Scopus (386) Google Scholar, 17Benaroudj N. Goldberg A.L. Nat. Cell Biol. 2000; 2: 833-839Crossref PubMed Scopus (119) Google Scholar). In addition, in the presence of ATP, PAN efficiently unfolds certain globular substrates and translocates them into the 20 S proteasome (17Benaroudj N. Goldberg A.L. Nat. Cell Biol. 2000; 2: 833-839Crossref PubMed Scopus (119) Google Scholar). PAN shows 40% homology to the six ATPases in the base of the 19 S particle and thus appears to have regulated proteasome function prior to the linkage of ubiquitination and proteolysis (11Zwickl P. Ng D. Woo K.M. Klenk H.P. Goldberg A.L. J. Biol. Chem. 1999; 274: 26008-26014Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Both proteasome-regulatory complexes are members of the AAA superfamily of multimeric ATPases, which also includes the ATP-dependent proteases Lon and FtsH and the regulatory components of the bacterial ATP-dependent proteases ClpAP, ClpXP, and HslUV (18Baumeister W. Walz J. Zuhl F. Seemuller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1295) Google Scholar, 19Larsen C.N. Finley D. Cell. 1997; 91: 431-434Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The unifying feature of the AAA superfamily is an ATPase of whose structural has been by x-ray D. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Huber R. Nature. 2000; PubMed Scopus Google Scholar, B. R. A. M. J. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). The AAA proteins a in this which contains the Walker A and B binding and In to ATP-dependent AAA proteins in from to F. M. 1995; PubMed Scopus Google Scholar, M. Cell Biol. 1998; 8: PubMed Google Scholar). the of the AAA their of is not well In the the ATPase and the conformational changes for their we on PAN, whose and for the of ATP in proteolysis A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 17Benaroudj N. Goldberg A.L. Nat. Cell Biol. 2000; 2: 833-839Crossref PubMed Scopus (119) Google Scholar). The degradation of a globular protein by the PAN-20S complex in (6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google ATPase association with the 20 S opening of the substrate entry in the 20 S binding of the substrate to the global unfolding of the substrate on the ATPase and translocation of the unfolded substrate into the 20 S particle. These functions by conformational transitions in the ATPase complex ATP hydrolysis to or of for ATP, and a of ATP-dependent proteolysis the of the structural changes associated with each of 19 S subunits have been by or is of ATP binding and hydrolysis the structure of the eukaryotic 19 S particle or the homologous PAN ATPase complex A. P. N. P. J. L. D. J. O. M. 2001; 2: PubMed Scopus Google Scholar, M. T. Biol. 2006; Scopus Google Scholar, N. Y. Glickman M.H. J. 2001; 20: PubMed Scopus Google Scholar, R. Tanaka K. Biochem. 2001; PubMed Scopus Google Scholar, M.H. Rubin D.M. O. G. Baumeister W. Finley D. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). To the of nucleotide binding and hydrolysis on the conformation of PAN, we its to proteases in the or presence of ATP, ATPγS ATP or and the by studies of the sensitivity of PAN to proteinase K distinct conformations to the and ADP-bound forms, conformational transitions to ATP hydrolysis. Using the structures of a homologous AAA we were to the of the PAN and the of conformational changes by PAN and were to from with as A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). that do not PAN. M. Smith and A. L. bind the 20 S proteasome (6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). of PAN was in the with or ATP, or and at 20 was at μm) or proteinase and were at 20 for were on and with was used as a substrate for degradation by PAN and 20 S proteasome, as A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). and protein were to a with and for degradation at the core was as with ATP, or were and was with These were and were ATPγS bound to PAN was from nucleotide a as Goldberg A.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). concentrations of PAN were at with a of from of PAN from the was as by protein and The of bound nucleotide was by the of the with the of bound to PAN. To the conformational transitions in PAN that from nucleotide binding and hydrolysis, five proteolytic were trypsin, and proteinase K not trypsin of PAN for 20 at 20 to the most and yielded distinct fragments of the and which when of trypsin to were not was its to the of fragments by of PAN with we were to the to we have in this on the by with To the of nucleotides on trypsin we the differed of PAN with ATP, or of nucleotides the of fragments they an of The of fragments in the presence or of a nucleotide differed in two the of each a of and the These changes in the of trypsin and thus conformational changes in PAN. To the of the binding for the changes in to trypsin, we PAN with concentrations of ATP, or ATPγS, by with trypsin and by The were and the which was in the presence of was by with ATP is in When the of this were nucleotide in each a was that with a binding of the nucleotide to by The concentrations of nucleotides that of were very for ATP ATPγS These changes in sensitivity were to nucleotide binding to PAN, and not to some on trypsin because in concentrations of ATP in the of hydrolysis by trypsin or the of the generated not Surprisingly, the of PAN for nucleotides μm) was much lower than the for the ATP-dependent breakdown of by the PAN-20S complex μm) J. J. 2000; PubMed Scopus Google or at Ng and A. L. To that the binding of nucleotides to PAN is on the Walker A and Walker B we two of PAN Science. 1999; PubMed Scopus Google Scholar, M. B. J. 1998; PubMed Scopus Google Scholar, K. M. R. J. Cell Biol. PubMed Scopus Google Scholar). we an for the in the Walker A which in ATPases is for nucleotide The of peptides generated of this Walker A with trypsin was by the presence of of the nucleotides Although was generated at in the presence of a this that a small of nucleotide binding in the also a for the in the Walker B motif, which is required for ATP hydrolysis in The Walker B in PAN the changes in trypsin sensitivity of ATP, or ATPγS as the PAN the conformational changes by the trypsin protection nucleotide binding at the Walker A motif, not ATP hydrolysis, as was also by the with and To that were affecting the of PAN to ATP-dependent protein degradation by the 20 S particle, we the degradation of an protein PAN catalyzed the ATP-dependent degradation of by the 20 as by the in protein the Walker A Walker B both conformational changes by trypsin and ATP-dependent proteolysis the at concentrations of the To the and the conformational changes in PAN nucleotide we used degradation to the generated by A was for each a small of the or in PAN were by trypsin, and all the fragments that were to trypsin an that were by the structure of the The and fragments, in the presence or of a lack the and The residues, which are were not in and to a with a a of was only in the presence of The and were of and are the of The in the presence of The location of the fragments and that conformational changes both the Walker A and B and the The of is in the of the Walker A and B and thus its by local structural changes nucleotide is from the of the Walker A and B and from the of binding of a nucleotide an in the of the PAN Although is that is adjacent to the Walker in the this that nucleotide binding has global on the structure of PAN. the of and overlap in with the the of in the presence of a nucleotide of in the of nucleotides generated of affecting the of and to generated by of a because the of with the of that and generated from than those that To the the and we the of each in by and the of generated the of ATP in the The of the of each of the fragments, the that fragments and are generated from the at the of each the at which this is to the of PAN for nucleotide PAN ATPγS suggest that PAN is a subunits The mechanism by which in the ring some subunits bound whereas To this we nucleotide binding to PAN ATPγS and to PAN from the of PAN, of were with PAN in the in the of this binding of four nucleotides PAN These were at a of ATPγS that binding not that each PAN a of four ATPγS saturating and the of conformations as by the trypsin fragments and ADP-bound of the and ADP-bound of PAN are to have (6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 7Benaroudj N. Zwickl P. Seemuller E. Baumeister W. Goldberg A.L. Mol. Cell. 2003; 11: 69-78Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), not in to trypsin, the changes associated with ATP hydrolysis not or a proteases to were in the generated binding of ATPγS and in the fragments generated by proteinase K in the presence of a not differed from those with by the 20 of PAN were proteinase K which is that this is the fragments generated with proteinase K to those with were not to the and as a in and not A of the fragments is in the The that two of conformational changes were by a in the of was not with whereas was an in was with The in the of was not with and the of was to with trypsin, the changes with proteinase K the including the The conformational to the of in the Walker A and B and to local structural changes associated with the of the the of a global conformational which to the very carboxyl of the These two a of conformational changes associated with ATP binding and hydrolysis to In this with trypsin and proteinase K have the structure of PAN and the of nucleotide binding and ATP hydrolysis. The trypsin is because that are conformations in the The overlap fragments and from distinct conformations of the that the and is in the presence or of a In the of a a of the B conformation to a which The of conformations in a complex is most to nucleotide binding to only some of six subunits of PAN. Partial occupancy of the by nucleotides was by a nucleotide binding which that only four ATPγS bound PAN at saturating The of conformations at the PAN are an for because the transitions of subunits conformations is to the ability of PAN to promote substrate unfolding and the most of is that a contains that do not bind nucleotide and the A conformation as well as that are to nucleotide and the B or of the trypsin fragments that distinct of PAN, each of bound and is also that in PAN of trypsin in of with the nucleotide binding or with distinct when of ATPγS bind to PAN. D. M. and A. L. is for a occupancy in AAA A of the AAA demonstrated that subunits are into with nucleotide binding and Cell. 2005; Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In addition, studies on ATP binding in the protease four nucleotides bound which at the was believed to Goldberg A.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Because is in a hexameric AAA are to the In addition, nucleotide occupancy has been in where the structure bound to only subunits of the M. Huber R. Nature. 2000; PubMed Scopus Google Scholar). appears most that some of the in a PAN bind nucleotide and conformational transitions whereas do of the peptides generated by trypsin and proteinase K several that are the structure of PAN. both proteases yielded a of fragments that the of PAN, that the of were the of PAN, which a (11Zwickl P. Ng D. Woo K.M. Klenk H.P. Goldberg A.L. J. Biol. Chem. 1999; 274: 26008-26014Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), were of protease or nucleotide that this structure nucleotide Although proteinase K has a much substrate than trypsin, both yielded fragments, that the to and are not to the of the and are fragments of and that were generated nucleotide The of the of fragments to the protease on the presence of a that function a adjacent that in their by both proteases and to function as a is and by the of and and the of and Although the with trypsin and proteinase K fragments and revealed some the conformational changes by trypsin are binding of whereas those by proteinase K to changes to ATP hydrolysis. Both PAN and the eukaryotic 19 S complex to a of changes in their on ATP binding and hydrolysis (6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The in the function of ATPases is the of a ring homologous AAA members as or nucleotide binding is an for ring M. G. A. Baumeister W. Goldberg A.L. Nat. Struct. Biol. 1997; PubMed Scopus Google Scholar). PAN, in the of nucleotide and the changes by trypsin probably to of into a A substrate In studies with the to PAN only when PAN was with A. A. and A. L. in These suggest that PAN a conformation for substrate binding when a ATP is bound. conformation is by proteinase K of PAN bound to ATPγS, where the and are whereas the central including the Walker A and B motif, The in the ATPase substrate unfolding, is to on ATP hydrolysis (6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 7Benaroudj N. Zwickl P. Seemuller E. Baumeister W. Goldberg A.L. Mol. Cell. 2003; 11: 69-78Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). the conformational changes by proteinase which the ATPγS and the bound forms, with changes in were to an for ATPγS proteinase K as we for nucleotide binding with is that this to the 300-500 for ATP of protein is to that the ATP hydrolysis was at or at whereas the protease sensitivity were at 20 and thus the of (Ka ∼ 20-40 μm) and (Km ∼ μm) for ATP that hydrolysis of ATP to a on ATP the of is than that of the of ATP the of and because ATP is not at 20 this is probably a of ATP the of These a for PAN function that by we the x-ray structures of AAA ATPase D. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, R. Nat. Struct. Biol. 1998; PubMed Scopus Google Scholar), A. B. E. P. J. G. M. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar), and M. Huber R. Nature. 2000; PubMed Scopus Google Scholar, J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 2000; Full Text Full Text PDF PubMed Scopus Google and homology in their and their homology structural of proteins a architecture of the to on which is the regulatory of the HslUV complex E. of the eukaryotic Although and PAN have in their and In PAN, the in the of the whereas in this is in Walker A and B The structure of that this not the Walker because the is of the Walker A and B that this from a that the M. Huber R. Nature. 2000; PubMed Scopus Google Scholar). To this we the homology PAN and and of the to the at the of the terminus. of the to the of PAN, the homology the from to studies of PAN in complex with the 20 S a ring an outer that to to the (6Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), and resembles of HslUV M. G. A. Baumeister W. Goldberg A.L. Nat. Struct. Biol. 1997; PubMed Scopus Google Scholar). The structure of PAN was and is with of protease Using the of PAN and we the on the structure of that probably to of protease sensitivity in PAN The two in PAN to nucleotide binding with trypsin were to the structure of HslU. In the presence of trypsin at to the structure of this in to the Walker A and B and the bound at most a local associated with nucleotide In the four of the six subunits are bound to a nucleotide M. Huber R. Nature. 2000; PubMed Scopus Google Scholar), as was for PAN. overlap in the fragments generated trypsin that the PAN ring also contains both bound and In to its to the Walker A and B is a location for a conformational to In a we have the in PAN to the and the conformational changes substrate binding and ATP hydrolysis are The by a at was only in the of a the of PAN, most that this in a that a to the When modeled the structure of this to a the and In and the structures of AAA the of the nucleotide binding of the in a the local conformational changes in this to in the Walker A and B of the whereas the global changes to in the of the structural of the In proteinase K the generated in the is in to is to ATP hydrolysis of nucleotide In this the at the base of a that lies the and in from the Walker A and B motif, is the in the to the in the we this as an of a conformational by ATP hydrolysis, the in the structure of that this a local from of the of ATP ATP hydrolysis. In studies that the proteasome-regulatory ATPase PAN distinct conformational changes nucleotide binding and ATP hydrolysis. Although the from of protease sensitivity in PAN are this are the structural to the PAN and in the of PAN that are in substrate unfolding, and translocation, and thus to a of the function of this proteasome-regulatory for in the protease protection for of of A.