AAA+ ATPase p97/VCP mutants and inhibitor binding disrupt inter-domain coupling and subsequent allosteric activation

Abstract

The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), (2) the interprotomer interactions (p97A232E), or (3) the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function. The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), (2) the interprotomer interactions (p97A232E), or (3) the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function. p97 is a member of the classic clade of AAA+ ATPases and is an essential cellular enzyme. p97 is composed of six protomers forming a homo-hexamer with an N-terminal domain (NTD) and two tandem ATPase domains D1 and D2 (1Erzberger J.P. Berger J.M. Evolutionary relationships and structural mechanisms of AAA+ proteins.Annu. Rev. Biophys. Biomol. Struct. 2006; 35: 93-114Crossref PubMed Scopus (566) Google Scholar). p97 is an essential protein in regulating cellular homeostasis from membrane fusion, Endoplasmic Reticulum-Associated Degradation, Mitochondrial-Associated Degradation (MAD), Chromatin-Associated Degradation to NF-κB activation (2Stach L. Freemont P.S. The AAA+ ATPase p97, a cellular multitool.Biochem. J. 2017; 474: 2953-2976Crossref PubMed Scopus (67) Google Scholar). A common thread among these applications is the recruitment of numerous cofactors to process ubiquitinated substrates, through the generation of mechanical energy from ATP hydrolysis. Critical functions for p97 include the translocation and restructuring of proteins from large cellular structures such as organelle membranes and extraction of ubiquitinated client proteins to facilitate their degradation through the ubiquitin-proteasome system. In this role, p97 recruits ubiquitin-binding cofactors to denature ubiquitinated substrates by pulling the polypeptide through the central pore (3Cooney I. Han H. Stewart M.G. Carson R.H. Hansen D.T. Iwasa J.H. Price J.C. Hill C.P. Shen P.S. Structure of the Cdc48 segregase in the act of unfolding an authentic substrate.Science. 2019; 365: 502-505Crossref PubMed Scopus (77) Google Scholar, 4Twomey E.C. Ji Z. Wales T.E. Bodnar N.O. Ficarro S.B. Marto J.A. Engen J.R. Rapoport T.A. Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding.Science. 2019; 365eaax1033Crossref PubMed Scopus (122) Google Scholar). Cryo-EM analyses of full-length p97 in the substrate-free form have established the overall organization of N, D1, and D2 domains (Fig. 1A) in three distinct quaternary conformations that correspond to distinct nucleotide occupancies of the D1 and D2 nucleotide-binding sites (Fig. 1B) (5Banerjee S. Bartesaghi A. Merk A. Rao P. Bulfer S.L. Yan Y. Green N. Mroczkowski B. Neitz R.J. Wipf P. Falconieri V. Deshaies R.J. Milne J.L.S. Huryn D. Arkin M. et al.2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.Science. 2016; 351: 871-875Crossref PubMed Scopus (221) Google Scholar). Conformation I is observed when ADP is bound to both the D1 and D2 domains, while conformation II is observed when ADP in the D2-binding site is exchanged with ATPγS, which is a slowly hydrolyzable ATP analog to preserve the ATP-bound structure for cryo-EM analysis. Relative to conformation I, there is a rotational twist of the D2 domain with relatively minimal changes in the D1 and N domains. Conformation III is observed when ATPγS is bound to both D1 and D2 nucleotide-binding sites and results in substantial tertiary and quaternary structural rearrangements in the D1 and N domains. The N-domain in conformation III adopts a distinct “up” position, enabling its binding to endogenous cofactors required for subsequent substrate binding and processing. The nucleotide occupancy in these three conformational states is consistent with the higher affinity of the D1 domain for both ADP and ATP ligands relative to the D2 domain, with a ∼40-fold lower KD in D1 relative to D2, as measured by Surface Plasmon Resonance (SPR) experiments (6Bulfer S.L. Chou T.F. Arkin M.R. P97 disease mutations modulate nucleotide-induced conformation to alter protein-protein interactions.ACS Chem. Biol. 2016; 11: 2112-2116Crossref PubMed Scopus (19) Google Scholar). All three conformations of substrate-free p97 display 6-fold symmetry. However, substrate binding results in the conversion of the hexameric arrangement to a spiral arrangement of the six protomers similar to the quaternary conformation observed for most other members of the AAA+ ATPase family (3Cooney I. Han H. Stewart M.G. Carson R.H. Hansen D.T. Iwasa J.H. Price J.C. Hill C.P. Shen P.S. Structure of the Cdc48 segregase in the act of unfolding an authentic substrate.Science. 2019; 365: 502-505Crossref PubMed Scopus (77) Google Scholar, 4Twomey E.C. Ji Z. Wales T.E. Bodnar N.O. Ficarro S.B. Marto J.A. Engen J.R. Rapoport T.A. Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding.Science. 2019; 365eaax1033Crossref PubMed Scopus (122) Google Scholar). Due to the essential role played by p97 in cellular processes, it is no surprise that a number of multisystem diseases are associated with mutations and dysfunctions in p97. Diseases arising from protein degradation defects (7Deshaies R.J. Proteotoxic crisis, the ubiquitin-proteasome system, and cancer therapy.BMC Biol. 2014; 12: 94Crossref PubMed Scopus (212) Google Scholar) and DNA repair (8Roux B. Vaganay C. Vargas J.D. Alexe G. Benaksas C. Pardieu B. Fenouille N. Ellegast J.M. Malolepsza E. Ling F. Sodaro G. Ross L. Pikman Y. Conway A.S. Tang Y. et al.Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.Sci. Transl. Med. 2021; 13eabg1168Crossref PubMed Scopus (8) Google Scholar) such as cancer, viral infections such as the poliovirus (9Arita M. Wakita T. Shimizu H. Valosin-Containing protein (VCP/p97) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection.J. Virol. 2012; 86: 5541-5553Crossref PubMed Scopus (49) Google Scholar), and even neurodegenerative disorders all implicate p97 as crucial in disease progression. p97 is therefore an attractive therapeutic target (10Huryn D.M. Kornfilt D.J.P. Wipf P. P97: An emerging target for cancer, neurodegenerative diseases, and viral Med. Chem. PubMed Scopus Google Scholar). The of p97 mutations are associated with a disease which is by a of and known as of the and J. S. D. A. associated with disease of and is by valosin-containing PubMed Scopus Google Scholar). these mutations to in the and of the p97 protein and the of p97 cofactors the of the protein, there is in the mechanism of mutants are also by an in ATPase H. C. Freemont P.S. The role of the N-domain in the of the ATPase Biol. Chem. 2012; PubMed Scopus Google Scholar) and higher substrate processing relative to p97 Deshaies R.J. A. mutations in binding and substrate 2019; PubMed Scopus Google Scholar), these have to mutations in the D2 domain of p97 have also in such as Amyotrophic Lateral Sclerosis J. M. Y. J.R. M. S. J. J. L. M. D. et mutations as a of PubMed Scopus Google Scholar), which to have and a in the mechanism of other mutations in the are implicated in cancer drug resistance P. F. T. Chou T.F. J. mutations in the of ATPase and resistance to 2017; PubMed Scopus Google Scholar), the role of p97 the and its in A central in the of the disease is the can to in protein structural on p97 mutants from studies of p97 D. is the basis for defects of p97 Biol. Chem. PubMed Scopus Google Scholar), of protein of full-length p97 mutants A basis for in disease mutants of 2016; PubMed Scopus Google Scholar), and cryo-EM of full-length p97 in complex with cofactors Deshaies R.J. A. mutations in binding and substrate 2019; PubMed Scopus Google Scholar, M. Y. H. Y. C. L. M. conformations of in the human p97 complex and the mechanism of a 2021; 12: PubMed Scopus Google Scholar). Here, we have full-length of p97 three for their relatively in the neurodegenerative for its relatively in the D2 domain in p97 and its role in and for its in resistance to the drug P. F. T. Chou T.F. J. mutations in the of ATPase and resistance to 2017; PubMed Scopus Google Scholar). Our experiments with these mutants to the to which the mutations the conformational p97 is to structural in both substrate-free and in the of bound protein substrates and we to substrate-free is to the of the mutations on quaternary studies have that the of mutations is in the between conformations on the domains (6Bulfer S.L. Chou T.F. Arkin M.R. P97 disease mutations modulate nucleotide-induced conformation to alter protein-protein interactions.ACS Chem. Biol. 2016; 11: 2112-2116Crossref PubMed Scopus (19) Google Scholar) or Deshaies R.J. A. mutations in binding and substrate 2019; PubMed Scopus Google Scholar). We also cryo-EM structures of full-length p97 to in the of ADP and ATPγS and the binding of the the overall quaternary arrangement of the N, D1, and D2 domains. results a of the structural mechanisms the of the disease mutations and an of the resistance of the to inhibition by All mutants relative ATPase similar to studies and H. C. Freemont P.S. The role of the N-domain in the of the ATPase Biol. Chem. 2012; PubMed Scopus Google Scholar, Deshaies R.J. A. mutations in binding and substrate 2019; PubMed Scopus Google Scholar, P. F. T. Chou T.F. J. mutations in the of ATPase and resistance to 2017; PubMed Scopus Google Scholar, D. is the basis for defects of p97 Biol. Chem. PubMed Scopus Google Scholar, D. An disease in binding and PubMed Scopus Google Scholar). The structural of the mutations on p97 structure as by cryo-EM studies two distinct the in relative to are observed in the of bound ADP ATPγS (Fig. or in the of bound ATPγS ADP (Fig. are in In the p97R191Q, p97R155H, and the in conformational as with is observed when the D1 and D2 domains are both by ADP (Fig. In these the conformation of the D1 domain of the structural observed ATPγS binding to that these mutations the conformational ATP is most in p97R191Q, with and similar in the D1 domain, of a and is the in the with from the domain D. L. L. D. A novel conformation in p97 by structures of J. PubMed Scopus Google Scholar) (Fig. to interactions in of the consistent with in this domain, observed as a in resolution in the N-domain cryo-EM is consistent with studies the N-domain between “up” and conformations in the is to the of the A basis for in disease mutants of 2016; PubMed Scopus Google Scholar). is the of the the N and D1 domains and by in (Fig. and the of the N-domain by an conformation in the A of the structural changes in the of is a of the the N and D1 domains an structure between and structural is observed in when ATPγS is bound to the D1 domain, which from changes the other of the to the D1 nucleotide-binding The from to an of by from to to the vital role plays in the in the N-domain of of the structure of in the a in the conformation of the between and of the D1 domain that is the between the N and D1 domains and The in the structure of this is such that it is to the conformation observed for this in the occupancy of the D1 nucleotide-binding site with is the between two p97 and the of the this in (Fig. the and of the N and D1 domains of the (Fig. for the of the N-domain and also results in a similar to that of in the between and of the D1 domain to the and a in an with Z. S. L. Y. M. S. Han T. Y. J. L. S. Ji Y. mutations in from a 2016; PubMed Scopus Google Scholar), the of this for the of p97. of in the as with all three mutants display similar structures when the D1 and D2 domains are bound to ATPγS and in the to conformation III of (Fig. (5Banerjee S. Bartesaghi A. Merk A. Rao P. Bulfer S.L. Yan Y. Green N. Mroczkowski B. Neitz R.J. Wipf P. Falconieri V. Deshaies R.J. Milne J.L.S. Huryn D. Arkin M. et al.2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.Science. 2016; 351: 871-875Crossref PubMed Scopus (221) Google Scholar). is and are all to in conformation no the therefore the mutations these sites are to affect the quaternary conformation The of the conformational is in the of the the of the N-domain results in the of by Å to a of the protein, from the In the and other mutants with similar have an on or substrate binding it is in to the site cofactors are to with p97 P. H. The of interactions and in the of the AAA+ ATPase 2017; PubMed Scopus Google Scholar). the to have relative to when bound to either or cofactors L. Bulfer S.L. V. L. M. Arkin M.R. Y. et with S. A. PubMed Scopus Google Scholar). In to the mutants in the and minimal structural are observed as with when the D1 and D2 domains are by ADP (Fig. However, when bound to ATPγS, in to the quaternary structural changes observed in the conformation in these two mutants is in which the N-terminal domain is in the in a most conformation II of and results that the mutations ATPγS binding in the D1 domain from the structural of N domain from the to “up” we the to have minimal of the is a between the D1 and D2 domains. the structural role of this in the of interactions by which the structural changes in the D2 domain are to the D1 is in the pore of the the (Fig. that interactions to the can also the coupling between these domains. observed the the and mutants in of their on ATPase (Fig. that ATPase in the D2 domain, by the for ATP binding to the D1 domain for D2 this across the is in the is a potent of p97 with an of J. B. S. S. B. J. E. F. A. S. A. F. et of a and of the p97 ATPase Med. Chem. PubMed Scopus Google Scholar). The binding site of to the D2 domain in a p97 to the D1 and D2 domains structure a resolution of Å T. D. basis of p97 inhibition by the 2019; PubMed Scopus Google Scholar). In to the of binding in the of full-length protein, we cryo-EM studies of bound to in the of either ADP or The cryo-EM structure of the complex an overall resolution Å of in the D2 The is in by a of interactions through its and central and interactions the The tertiary and quaternary structures of the D1 and D2 domains the both that when is bound to the D2 domain, the conformation of the D1 domain is and to either the or the of the bound the N-domain is in the when either ADP or ATPγS the D1 nucleotide-binding site and the of p97 bound to in the of D1 nucleotide occupancy and this is observed in the structures in the the that the D2 domain is in conformation I. In this the of binding is similar to that observed for the and the conformation of the N and D1 domains is with binding of either ADP or ATPγS (Fig. However, these is in conformation I and the tertiary structure in the D2 domain as that seen for p97. CB-5083, as an inhibitor, can its on p97 by the allosteric conformational changes required for from conformation I to conformation II (Fig. The is by the allosteric which p97 to the nucleotide binding prevents the to conformation II of a of the conformational in the D2 domain by the bound (5Banerjee S. Bartesaghi A. Merk A. Rao P. Bulfer S.L. Yan Y. Green N. Mroczkowski B. Neitz R.J. Wipf P. Falconieri V. Deshaies R.J. Milne J.L.S. Huryn D. Arkin M. et al.2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.Science. 2016; 351: 871-875Crossref PubMed Scopus (221) Google Scholar). A of the interactions in the nucleotide-binding an for the observed with in the interactions of the two in ADP are to a nucleotide-binding domain, when ATPγS is the is involved in interactions with and in the interactions ATPγS binding the of quaternary structural changes interactions the binding site with the nucleotide-binding site of the D2 domain and interactions with of the that with the between and the of ATP is in the the generation of the of quaternary conformational changes seen ATPγS binding to The cryo-EM structural studies to a structural for the observed in ATPase of p97 Our results demonstrate that the of the mutations are through the of the N domain in the either through a of interactions in and or through in We that the of the mutants on p97 is a in recruitment to the in the conformational the N domain “up” is by and A basis for in disease mutants of 2016; PubMed Scopus Google Scholar, V. Deshaies R.J. A of is by a that multisystem S. A. 2017; PubMed Scopus Google Scholar) that these mutants an affinity for which bound p97 in the “up” and and a affinity for which p97 in the therefore these mutants to affect a of to the of the diseases, a of p97, in to I. G. D.T. of p97 in results in Biophys. PubMed Scopus Google Scholar). the of both and p97 ATPase in the D2 domain D. is the basis for defects of p97 Biol. Chem. PubMed Scopus Google Scholar, C. ATPase of protein D2 the and D1 to the Biol. Chem. PubMed Scopus Google Scholar), the of D2 ATP is on the of nucleotide in the D1 domain H. C. Freemont P.S. The role of the N-domain in the of the ATPase Biol. Chem. 2012; PubMed Scopus Google Scholar), a path of of nucleotide between D1 and D2 domains. However, we have observed no in the nucleotide-binding sites of the D2 domain in the the observed in ADP (6Bulfer S.L. Chou T.F. Arkin M.R. P97 disease mutations modulate nucleotide-induced conformation to alter protein-protein interactions.ACS Chem. Biol. 2016; 11: 2112-2116Crossref PubMed Scopus (19) Google Scholar) and ATPase to a conformational in the D2 nucleotide-binding a an in the D1 domain of the p97 facilitate a ATP in the D2 domain through this of the structure bound to either ATPγS (Fig. or in the D2 domain to show no in the binding of either nucleotide or drug relative to that the resistance by this to the changes in binding its changes in the conformation by the D2 domain from the D1 domain, a mechanism for p97 through a in the to the observed in ATPase P. F. T. Chou T.F. J. mutations in the of ATPase and resistance to 2017; PubMed Scopus Google Scholar) (Fig. and it is that the for nucleotide binding in the D1 domain for ATP in the D2 domain in is by the the D2 domain to through of D1 nucleotide it that the is to D2 to the D1 domain an pathway G. C. G. mechanism for the hexameric ATPase S. A. 2012; PubMed Scopus Google Scholar). Our studies of that this is in the of interactions by which the structural changes in the D2 domain are to the D1 by the to of D2 to the with the mutants to in a similar of conformational this to to an on the of ATP D. An disease in binding and PubMed Scopus Google Scholar). that the D2 domain, of on the of the nucleotide of the D1 domain, the of the in regulating D2 ATP hydrolysis. Our results that between D1 and D2 domains can by interactions to the domain allosteric structural studies with demonstrate that this with ATP binding to the D2 domain, the of binding is to the allosteric changes that and in a similar to that observed with such as (5Banerjee S. Bartesaghi A. Merk A. Rao P. Bulfer S.L. Yan Y. Green N. Mroczkowski B. Neitz R.J. Wipf P. Falconieri V. Deshaies R.J. Milne J.L.S. Huryn D. Arkin M. et al.2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.Science. 2016; 351: 871-875Crossref PubMed Scopus (221) Google Scholar) that ADP binding to either D1 or D2 also p97 in conformation I as observed with the of ATPγS in the D1 domain, it is for the D2 domain to the active conformation II the D1 domain can to the N-domain “up” conformation that while nucleotide from D1 to D2 D2 ATPase in the from D2 to D1 of nucleotide also the N-domain conformation and therefore conformational binding in conformation I in to we in the the conformation that the from conformation I to II is to to conformation the of the structures of p97 disease mutants and of the structure of bound to the of and interactions that p97 (Fig. The of resolution for disease mutants and of the and in allosteric changes by mutations binding a for drug design of potent can for regulating p97 function. in disease for of the for of the human p97 mutants with as in D. A. M. design of with 2019; 35: PubMed Scopus (19) Google Scholar). with E. p97 in of and and to the to a of and a and with The protein in and in a with the of of the and to a in of to and to The and for the to and the measured for The ATPase the as in S. B. J. S. J. B. E. S. A. F. M. et al.Targeting the ATPase p97 as an to cancer through of protein PubMed Scopus Google Scholar). The protein to p97 with ATP in in of and for the to for and of to the and the The for of and for The across the three and as In of the for and the to a of in protein with 1 and 1 ADP or 1 ATPγS or 1 on the and on the on or for and on a of in the to with of protein on the The for by in in in of protein with between and with a of on either a or with a or to 1 for a of the cryo-EM of Cryo-EM and and processing to to to to to to to to to to to to resolution resolution and in a In all processing in or A. for cryo-EM structure 2017; PubMed Scopus Google Scholar) in in Å as and extraction in to and with and all the p97 or p97 bound with and of the to the a of of and in Biol. PubMed Scopus Google Scholar). to for a and for of the cryo-EM structure and analysis. The cryo-EM structures in the ADP or ATPγS bound states (5Banerjee S. Bartesaghi A. Merk A. Rao P. Bulfer S.L. Yan Y. Green N. Mroczkowski B. Neitz R.J. Wipf P. Falconieri V. Deshaies R.J. Milne J.L.S. Huryn D. Arkin M. et al.2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.Science. 2016; 351: 871-875Crossref PubMed Scopus (221) Google Scholar) to for the and mutants D.M. E.C. T.E. a for and Chem. PubMed Scopus Google Scholar), the the to an structure for of the in D. G. B. S. M.G. R.J. et structure and in Struct. Biol. 2019; PubMed Scopus Google Scholar). The the in and E.C. J.H. T.E. Structure for and 2021; PubMed Scopus Google Scholar). The and have in the with and and in the with of and for p97, and for p97 bound to and All other are from the H. C. Freemont P.S. The role of the N-domain in the of the ATPase Biol. Chem. 2012; PubMed Scopus Google Scholar, P. F. T. Chou T.F. J. mutations in the of ATPase and resistance to 2017; PubMed Scopus Google Scholar, D. An disease in binding and PubMed Scopus Google Scholar, and from a PubMed Scopus Google Scholar). S. S. is and of a drug in All other that have no of with the of this We members of the for We for with and in the of the A of this by and the and through a of by the of and B. and S. S. B. A. and S. C. B. C. B. A. and S. C. B. A. S. and S. S. B. C. B. A. and S. S. B. A. S. and S. S. B. A. S. and S. S. and by to S. S. from a the for and the The is the of the and the of the of

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