Analysis of Nucleotide Binding to P97 Reveals the Properties of a Tandem AAA Hexameric ATPase

Abstract

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5′-O-(thiotriphosphate) (ATPγS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPγS can saturate all 6 nucleotide binding sites in D1, only 3–4 of the 6 D2 sites can bind ATPγS simultaneously. ATPγS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis. p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5′-O-(thiotriphosphate) (ATPγS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPγS can saturate all 6 nucleotide binding sites in D1, only 3–4 of the 6 D2 sites can bind ATPγS simultaneously. ATPγS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis. The “ATPases associated with various cellular activities” (AAA+) 3The abbreviations used are: AAA, ATPases associated with various cellular activities; ITC, isothermal titration calorimetry; B-ADP, BODIPY ADP; NSF, N-ethylmaleimide-sensitive factor; SRH, second region of homology; HPLC, high pressure liquid chromatography; PDB, Protein Data Bank; ATPγS, adenosine 5′-O-(thiotriphosphate); AMPPNP, 5′-adenylyl-β,γ-imidodiphosphate; ADP-AIF3, adenosine 5′-diphosphate-aluminum fluoride. 3The abbreviations used are: AAA, ATPases associated with various cellular activities; ITC, isothermal titration calorimetry; B-ADP, BODIPY ADP; NSF, N-ethylmaleimide-sensitive factor; SRH, second region of homology; HPLC, high pressure liquid chromatography; PDB, Protein Data Bank; ATPγS, adenosine 5′-O-(thiotriphosphate); AMPPNP, 5′-adenylyl-β,γ-imidodiphosphate; ADP-AIF3, adenosine 5′-diphosphate-aluminum fluoride. superfamily of proteins utilize ATP binding and hydrolysis to induce conformational change. This conformational change provides mechanical energy used in diverse functions to remodel substrate proteins and nucleic acids (1Hanson P.I. Whiteheart S.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 519-529Crossref PubMed Scopus (887) Google Scholar). AAA+ ATPases are defined by the presence of one or two AAA domains, and many form homohexameric rings. The enzymatic mechanisms of the more complex AAA+ ATPases have remained largely unclear, especially those like p97, which contain two AAA domains and thus 12 ATPase active sites within a functional hexamer. p97 (also known as vasolin-containing protein (VCP) in mammals and Cdc48p in yeast) is an abundant and essential eukaryotic AAA-ATPase that remodels substrate protein complexes and is best characterized in endoplasmic reticulum-associated degradation and homotypic membrane fusion events of organelle biogenesis following mitosis (2Ye Y. J. Struct. Biol. 2006; 156: 29-40Crossref PubMed Scopus (173) Google Scholar, 3Uchiyama K. Kondo H. J Biochem. (Tokyo). 2005; 137: 115-119Crossref PubMed Scopus (90) Google Scholar). A proposed Cdc48p segregase activity has been demonstrated in vitro for a membrane-tethered transcription factor (activated by a mechanism that co-opts the later stages of endoplasmic reticulum-associated degradation), which, in the presence of Cdc48p and Ufd1p-Npl4p adaptor, was separated from a polyubiquitinated membrane anchor (4Shcherbik N. Haines D.S. Mol. Cell. 2007; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Although the detailed mechanism by which p97 carries out remodeling activity remains obscure, it involves the formation of multiple p97-adaptor complexes and conformational change during the ATPase hydrolysis cycle (2Ye Y. J. Struct. Biol. 2006; 156: 29-40Crossref PubMed Scopus (173) Google Scholar, 5Dreveny I. Pye V.E. Beuron F. Briggs L.C. Isaacson R.L. Matthews S.J. McKeown C. Yuan X. Zhang X. Freemont P.S. Biochem. Soc. Trans. 2004; 32: 715-720Crossref PubMed Scopus (72) Google Scholar, 6Pye V.E. Dreveny I. Briggs L.C. Sands C. Beuron F. Zhang X. Freemont P.S. J. Struct. Biol. 2006; 156: 12-28Crossref PubMed Scopus (84) Google Scholar, 7Beuron F. Dreveny I. Yuan X. Pye V.E. McKeown C. Briggs L.C. Cliff M.J. Kaneko Y. Wallis R. Isaacson R.L. Ladbury J.E. Matthews S.J. Kondo H. Zhang X. Freemont P.S. EMBO J. 2006; 25: 1967-1976Crossref PubMed Scopus (90) Google Scholar, 8Rouiller I. DeLaBarre B. May A.P. Weis W.I. Brunger A.T. Milligan R.A. Wilson-Kubalek E.M. Nat. Struct. Biol. 2002; 9: 950-957Crossref PubMed Scopus (184) Google Scholar, 9Rouiller I. Butel V.M. Latterich M. Milligan R.A. Wilson-Kubalek E.M. Mol. Cell. 2000; 6: 1485-1490Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). p97 consists of three domains: an N domain that interacts with many proteins and two homologous AAA domains with ATPase activity, D1 and D2. The AAA domains contain conserved motifs necessary for nucleotide binding (Walker A) and hydrolysis (Walker B), and mutation of these motifs may be used to specifically decrease these functions (10Ogura T. Wilkinson A.J. Genes Cells. 2001; 6: 575-597Crossref PubMed Scopus Google Scholar). p97 has a stable homohexameric with the D1 and the D2 domains two a and N domains from the D1 domains V.E. Dreveny I. Briggs L.C. Sands C. Beuron F. Zhang X. Freemont P.S. J. Struct. Biol. 2006; 156: 12-28Crossref PubMed Scopus (84) Google Scholar). The N domain proteins as and that the ATP hydrolysis cycle of of F. Dreveny I. Yuan X. Pye V.E. McKeown C. Briggs L.C. Cliff M.J. Kaneko Y. Wallis R. Isaacson R.L. Ladbury J.E. Matthews S.J. Kondo H. Zhang X. Freemont P.S. EMBO J. 2006; 25: 1967-1976Crossref PubMed Scopus (90) Google Scholar). of conformational from ATP binding and hydrolysis has been proposed two within a the D1, and D2 domains conformational change domains B. Zhang X. J. Struct. Biol. 2004; PubMed Scopus Google Scholar). AAA domains within a AAA are by two arginine within the second region of that may the active T. Whiteheart S.W. Wilkinson A.J. J. Struct. Biol. 2004; PubMed Scopus Google Scholar). are conserved in p97 and are necessary for ATP hydrolysis C. R. Zhang X. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of the p97 functions the ATP hydrolysis cycle has been from and all of p97 D1 to D2 has been to an ATP a and with nucleotide B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar, B. Brunger A.T. Nat. Struct. Biol. PubMed Scopus Google Scholar, T. Pye V.E. Briggs L.C. Beuron F. Kondo H. J. Zhang X. Freemont P.S. J. Struct. Biol. PubMed Scopus Google Scholar, X. B. Kondo H. J. H. M. Freemont P.S. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, I. Kondo H. K. Zhang X. Freemont P.S. EMBO J. 2004; PubMed Scopus Google Scholar). these ADP has been to p97 H. May A.P. Weis W.I. 2005; Full Text Full Text PDF PubMed Scopus Google and ADP and ATP have been to bind to D2 C. X. J. Biol. Full Text Full Text PDF PubMed Scopus (72) Google Scholar). is that ATP may be by all sites in the D2 as ATP to a p97 R. Biochem. J. PubMed Scopus Google the of p97 with to D2 of the in only one of three in the B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar). the mechanism of p97, have out a of the nucleotide binding to p97 D1 and D2 of the A to nucleotide binding to D1 and D2 have the and of ADP and ATP (ATPγS) binding to D1 and D2 detailed measurements have a conformational change of p97 ATPγS binding to D2. these the mechanism of and Protein p97 with an was and to the A and and the and by the at in the a with and by The and of was by and The of p97 was by at with as H. May A.P. Weis W.I. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). was separated from p97 by a membrane during the to of binding out in the following and all of p97 are of of by nucleotide was by of p97 at The protein was by the was and the was The of nucleotide was from at and the of nucleotide was as the of nucleotide to p97 in the for and the of than and associated with ATPγS binding of p97 binding to The are to a that p97 is at ATPγS in which ATPγS was to p97 ADP or by that ATPγS binding to p97 is at of nucleotide and is of ADP to of type and A in to ATPγS binding that the change from ATPγS binding to D2 and is by nucleotide to to a cooperative binding than of to a that ATPγS is with similar affinity to measurements of of binding to ATPγS that the of ATPγS binding is of ATPγS similar to The of ATPγS by p97 was by of p97 with a of ATPγS and to of the Protein and and the of nucleotide in the was The of ATPγS to be to the of was from the nucleotide the of nucleotide to than used as the nucleotide measurements by measurements at with a and used ADP in a used a of ADP or and p97, p97, and for to for The at of the ADP was and the of and ADP for p97 by the following from Y. Biochem. PubMed Scopus Google measurements by ATPγS to of p97 in a p97 was used for of as ATPγS to a or cooperative binding 2002; PubMed Scopus Google and of multiple are in titration measurements with a at nucleotide Data and to one and two of binding and two Biochem. 2000; PubMed Scopus Google Scholar, T. Biochem. PubMed Scopus Google Scholar). the of ADP by more complex binding and to and binding with higher of at least three of and the and are in of A and to nucleotide binding to p97 by the D1 and D2 domains nucleotide binding affinity of AAA domains, the A that interacts with nucleotide X. B. Kondo H. J. H. M. Freemont P.S. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google to mutation of the A was in D1 and D2 both and the an arginine in the was to in both D1 and D2 D1 at that this arginine the active of the of these to p97 the these ATPase activity to of p97 the A mutation to the D1 domain a decrease in ATPase activity, p97 in the D2 domain ATPase activity with type C. R. Zhang X. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). This is with the that the D2 domain is the of ATPase activity C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, C. Biochem. PubMed Scopus Google Scholar). of ADP to that ADP is to of p97 B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar, B. Brunger A.T. Nat. Struct. Biol. PubMed Scopus Google Scholar, T. Pye V.E. Briggs L.C. Beuron F. Kondo H. J. Zhang X. Freemont P.S. J. Struct. Biol. 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Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). the of p97 that ADP is to of D1 domains ADP these that ADP binds with affinity to D1 it is the and of the affinity of ADP for D1, out binding as a p97 was to B-ADP, and binding was by the of with D1 and D2 A to which domain binds The D2 similar binding to the that bind to D2. of D1 and binding necessary for thus that binds to D1 The of ADP binding to D1 was by the of at of the of the ADP the of ADP to be at ADP binding to p97 was by We an of ADP p97, an binding of a binding with the binding at higher ADP that ADP binds to a second have demonstrated that D2 can bind ADP B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar, C. X. J. Biol. Full Text Full Text PDF PubMed Scopus (72) Google in with a binding the The a higher affinity with a of and of and a affinity with a of but defined to the affinity of this was to for The of the high affinity is with the of a of D1 domains, as by and the is the as for D1 these that ADP binds with higher affinity to D1 than D2 We ADP to D1 the binding of ADP to D2. p97 was with for to of ADP from D1 H. May A.P. Weis W.I. 2005; Full Text Full Text PDF PubMed Scopus Google and ADP binding was by to higher of ADP binding to D1 and D2 ADP binding and remained similar to p97 these ADP binding to D2 is by ADP to the of ADP binding to D2 D1 ADP binding was was in the as D2 in p97 as by have ADP to the D1 domain of p97 with B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar, B. Brunger A.T. Nat. Struct. Biol. PubMed Scopus Google Scholar, T. Pye V.E. Briggs L.C. Beuron F. Kondo H. J. Zhang X. Freemont P.S. J. Struct. Biol. PubMed Scopus Google Scholar, X. B. Kondo H. J. H. M. 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Weis W.I. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). to D1, the D2 has affinity for a as the of ATP hydrolysis. of ATPγS to D1 and to ATP binding to as ATPase activity in p97 to out of ATP hydrolysis used a ATP ATP AMPPNP, has affinity for p97 than ATP R. Biochem. J. PubMed Scopus Google that for the of binding it that ATPγS to p97 with higher affinity than and used ATPγS to these and ADP from D1 by as the the of p97 have characterized ADP to p97, it to be for in We first ATPγS binding to D2 A which is in D2 nucleotide binding and has ADP to D1 The of the binding that a of D1 sites D1 sites is with ADP and binds to ATPγS in the The to a that with ADP be A that of ADP are present to the This that ATPγS binds to D1 with a of and an of of ATPγS ATPγS The of ADP for D1 by the is in with measurements of ADP binding to type and We ATPγS binding to D2 the D1 A which has affinity for nucleotide in D1 and contains ADP of a to binding a high affinity with a of and a affinity with a of and best the of ATPγS binding the high affinity was a of We the high affinity as ATPγS binding to D2 mutation of D2 high affinity ATPγS binding The affinity be a of affinity D2 domains, or more binding of ATPγS to the D1 to the of ADP binding to D1 and D2, ATPγS binding to A that D1 and D2 have similar for ATPγS (D1 and D2 are with measurements of the affinity of ADP binding and that ATPγS binding to D1 the affinity of ADP to D1 of ATPγS to ATPγS binding to p97, out The binding of ATPγS p97 that ATPγS binding was at ATPγS but at higher ATPγS The of the a of ATPγS binding p97 This is with ATPγS binding to sites from the D2 and the of D1 The later to ATPγS with ADP for binding sites in the D1 and the in of ADP and ATPγS ADP was a that for of ADP but that D1 and D2 have the affinity for ATPγS the of ADP at and that affinity ADP binding to D2 was The the of ATPγS to be and the of ATPγS was ATPγS We this by the of ATPγS by p97 a of ATPγS was to p97, and the of ATPγS was the was of ATPγS was to as at higher to p97 was to of nucleotide p97 this p97 a similar that the of ATPγS is by the presence of ADP in these that within a p97 only the 12 binding that the D1 and D2 have V.E. Dreveny I. Briggs L.C. Sands C. Beuron F. Zhang X. Freemont P.S. J. Struct. Biol. 2006; 156: 12-28Crossref PubMed Scopus (84) Google and that of D1 B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar, B. Brunger A.T. Nat. Struct. Biol. PubMed Scopus Google Scholar, T. Pye V.E. Briggs L.C. Beuron F. Kondo H. J. Zhang X. Freemont P.S. J. Struct. Biol. PubMed Scopus Google Scholar, X. B. Kondo H. J. H. M. Freemont P.S. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, I. Kondo H. K. Zhang X. Freemont P.S. EMBO J. 2004; PubMed Scopus Google but of D2 in the of B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google that one is by ATPγS, the is The of that the D1 is with 6 of ATPγS, the D2 can only bind 3–4 of with ATPγS to the mechanism of ATPγS binding to the D2 by a the D1 and D2 and to the D2 active The of this is to with D2 nucleotide binding C. X. J. Biol. Full Text Full Text PDF PubMed Scopus (72) Google to a change in the of the from the of nucleotide to or a conformational change in of ATPγS p97 a in that to a cooperative binding with a of and 2002; PubMed Scopus Google Scholar). We the titration with the D1 and D2 A to that this change was a of ATPγS binding to D2. the ATPγS mutation of D2 and was to in This that in are associated with ATPγS binding to the D2 The in within the ATPγS binding to D2. The of that three or more p97 ATPγS binding to the D2 A similar was in p97 from in this of at least p97 and Although the in the in and p97 is unclear, both suggest a cooperative change at least 3–4 We the nucleotide of D1 the cooperative by ATPγS D1 is in We of p97 ADP to to the D1 (D1 and affinity for and and p97 (D1 and ATPγS binding to both The three similar that the of D1 the cooperative associated with ATPγS binding to D2. of ATPγS it has been proposed that the activity of in a are by arginine fingers the from one to the C. R. Zhang X. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google these arginine in p97 are in the cooperative ATPγS ATPγS binding to the arginine in a decrease in the of the change from in type to in and a of the of the binding to a that ATPγS binds with a of to This is with measurements for p97 and that these the affinity of ATPγS binding The in of to can be to nucleotide binding the cooperative to with p97 is with nucleotide binding and an cooperative conformational change. We used to the of ATPγS binding to a to the to for ADP The of ATPγS binding from this was similar to that for p97 The for ATPγS binding to D1 and D2 was but of a similar to p97, and the of the was more which to conformational within the that mutation of this arginine the affinity or of ATPγS but to a of The for p97 to be associated with a conformational change that downstream of ATPγS and is by the arginine of arginine fingers of the are associated with of ATPase activity C. R. Zhang X. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google that this cooperative conformational change is for ATP hydrolysis. This the first quantitative study of the nucleotide binding of both D1 and D2 of a AAA ATPase and the homologous AAA domains of have of ADP binding to the D1 and D2 for D1 and for the D2 in the affinity of ATPγS binding to D1 and D2 is similar for both with a of has that the D2 is the of ATP hydrolysis C. J. Biol. Full Text Full Text PDF PubMed Scopus Google a by that D2 in multiple but D1 only to ADP B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar, B. Brunger A.T. Nat. Struct. Biol. PubMed Scopus Google Scholar, T. Pye V.E. Briggs L.C. Beuron F. Kondo H. J. Zhang X. Freemont P.S. J. Struct. Biol. PubMed Scopus Google Scholar). these and suggest that the in affinity of ADP for D1 and D2 provides a explanation for these Although the affinity of ATPγS for D1 and D2 is that the of binding is ATPγS binding is to a of D2 domains out of 6 the D1 binds ATPγS to The of ATPγS binding to D2 that ATPγS binding to the first 3–4 D2 sites to in the D2 sites to ATPγS and of ATPγS binding to D2 with The of ATPγS in D2 is in with of ATP to p97 R. Biochem. J. PubMed Scopus Google and only one D2 domain of the three in the of the of B. Brunger A.T. J. Mol. Biol. 2005; PubMed Scopus Google Scholar). the AAA+ ATPase is a for of ATP in homohexameric AAA proteins with as and H. J. Mol. Biol. 2001; PubMed Scopus Google Scholar, M. C. R. 2000; PubMed Scopus Google Scholar, Cell. 2005; Full Text Full Text PDF PubMed Scopus (141) Google but this is the first report for a AAA protein and a of mechanism the of AAA+ ATPase We have in a of the of one the affinity of the that ADP and ATPγS binding in D2 is by the presence of ADP in D1 and the affinity of ADP to the of ATPγS binding to D2 is by ADP in D1, and conformational in D2 are the of the of the D1 the first that the nucleotide binding of D1 and D2 are This out an of D1 and D2 in of as the detailed of ATP hydrolysis or in the of conformational energy to the of the out both can in of nucleotide this out a detailed analysis of to from nucleotide binding C. X. J. Biol. Full Text Full Text PDF PubMed Scopus (72) Google and have a cooperative conformational change associated with ATPγS binding to D2 in both and The of the conformational change that this involves at least three monomers, and have that the conserved arginine of the are to this change. this cooperative conformational change ATPγS binding that mutation of the but ATPγS binding affinity or this mutation decrease ATP hydrolysis to in with C. R. Zhang X. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google that the cooperative conformational change be an essential of the ATP hydrolysis of have that the by these to the ATP hydrolysis C. R. Zhang X. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of the D1 domain the active of the X. B. Kondo H. J. H. M. Freemont P.S. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google to conformational change. p97 is to or protein complexes by ATP binding and hydrolysis to change in nucleotide binding V.E. Dreveny I. 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Freemont Kondo H. J. Cell Biol. 2002; PubMed Scopus Google is of detailed of the of a 3–4 ATP binding and of at least three that the D2 of the p97 a mechanism in which all D2 sites bind and change (10Ogura T. Wilkinson A.J. Genes Cells. 2001; 6: 575-597Crossref PubMed Scopus Google Scholar). the and are more with a mechanism multiple of ATP hydrolysis and conformational change form a are to ATP binding and hydrolysis a and mechanism or are a as has been for Cell. 2005; Full Text Full Text PDF PubMed Scopus (141) Google Scholar). with AAA+ suggest that the D1 and D2 is similar to that of N-ethylmaleimide-sensitive factor The of ADP to the D1 domain of p97 one AAA of which binds ATP with high affinity and has only been to ATP or ATP Whiteheart S.W. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Whiteheart S.W. Weis W.I. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, P.I. R. Brunger A.T. Nat. Struct. Biol. PubMed Scopus Google Scholar). with the D2 of p97, the AAA of higher ATPase activity M. M. J. Biol. Full Text PDF PubMed Google Scholar, S.W. K. M. R. J.E. J. Cell Biol. PubMed Scopus Google Scholar). AAA+ ATP hydrolysis in one is by the of the EMBO J. 2002; PubMed Scopus Google and substrate remodeling activity is to hydrolysis at AAA domains J. M. Nat. Struct. Mol. Biol. 2007; PubMed Scopus Google Scholar). Although suggest that nucleotide binding to the D1 and D2 of p97 is out more complex of ATP hydrolysis in the presence of proteins similar to the p97 D2 to a with a AAA which binds 3–4 ATP in a similar of affinity with for D2 binding and a cooperative change ATP binding Cell. 2005; Full Text Full Text PDF PubMed Scopus (141) Google Scholar). these suggest that the presence of an AAA in p97, with and functional of the ATP hydrolysis mechanism of homohexameric AAA+ proteins may be We are to Dreveny and Pye for and for the We and McKeown for and for of with