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trans-Acting Arginine Residues in the AAA+ Chaperone ClpB Allosterically Regulate the Activity through Inter- and Intradomain Communication*

  • Cathleen Zeymer
    Affiliations
    Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
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  • Sebastian Fischer
    Affiliations
    Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
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  • Jochen Reinstein
    Correspondence
    To whom correspondence should be addressed: Dept. of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Tel.: 49-6221-486-502; Fax: 49-6221-486-585
    Affiliations
    Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany
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  • Author Footnotes
    * This work was supported by the Max Planck Society and by a Ph.D. scholarship from the German National Academic Foundation (to C. Z.).
Open AccessPublished:September 24, 2014DOI:https://doi.org/10.1074/jbc.M114.608828
      The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine. Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which also harbor the catalytic sites. Several AAA+ proteins, including ClpB/Hsp104, possess a pair of such trans-acting arginines in the N-terminal nucleotide binding domain (NBD1), both of which were shown to be crucial for oligomerization and ATPase activity. Here, we present a mechanistic study elucidating the role of this conserved arginine pair. First, we found that the arginines couple nucleotide binding to oligomerization of NBD1, which is essential for the activity. Next, we designed a set of covalently linked, dimeric ClpB NBD1 variants, carrying single subunits deficient in either ATP binding or hydrolysis, to study allosteric regulation and intersubunit communication. Using this well defined environment of site-specifically modified, cross-linked AAA+ domains, we found that the conserved arginine pair mediates the cooperativity of ATP binding and hydrolysis in an allosteric fashion.

      Introduction

      The molecular disaggregation machine ClpB/Hsp104 (caseinolytic peptidase B/heat shock protein 104) is crucial for maintaining protein homeostasis because it reactivates aggregated proteins under cellular stress conditions in concert with the DnaK/Hsp70 chaperone system (
      • Sanchez Y.
      • Lindquist S.L.
      HSP104 required for induced thermotolerance.
      ,
      • Doyle S.M.
      • Wickner S.
      Hsp104 and ClpB: protein disaggregating machines.
      ,
      • Hodson S.
      • Marshall J.J.
      • Burston S.G.
      Mapping the road to recovery: the ClpB/Hsp104 molecular chaperone.
      ,
      • Zolkiewski M.
      • Zhang T.
      • Nagy M.
      Aggregate reactivation mediated by the Hsp100 chaperones.
      ). Belonging to the superfamily of AAA+ proteins (ATPases associated with various cellular activities), ClpB/Hsp104 functions as a hexameric complex that converts the chemical energy from ATP hydrolysis into mechanical force (
      • Tucker P.A.
      • Sallai L.
      The AAA+ superfamily: a myriad of motions.
      ). Protein disaggregation by ClpB/Hsp104 involves the threading of single polypeptide chains out of the aggregate through the central pore of the hexameric ring (
      • Weibezahn J.
      • Tessarz P.
      • Schlieker C.
      • Zahn R.
      • Maglica Z.
      • Lee S.
      • Zentgraf H.
      • Weber-Ban E.U.
      • Dougan D.A.
      • Tsai F.T.
      • Mogk A.
      • Bukau B.
      Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB.
      ). High-resolution structural information is available for ClpB from Thermus thermophilus, showing a domain architecture that consists of a small N-terminal domain and two highly conserved AAA+ domains, also called nucleotide binding domains (NBD1
      The abbreviations used are: NBD1
      nucleotide binding domain 1
      NBD2
      nucleotide binding domain 2
      DLS
      dynamic light scattering
      MANT-dADP
      2′-deoxy-3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate
      NBD1-M
      ClpB construct comprising residues 141–534
      SLS
      static light scattering.
      and NBD2), per monomer (
      • Lee S.
      • Sowa M.E.
      • Watanabe Y.-H.
      • Sigler P.B.
      • Chiu W.
      • Yoshida M.
      • Tsai F.T.F.
      The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state.
      ). There is a long helical insertion into NBD1, named the M-domain, which was recently identified as a major regulatory element as well as the interaction site for the co-chaperone DnaK/Hsp70 (
      • Watanabe Y.H.
      • Takano M.
      • Yoshida M.
      ATP binding to nucleotide binding domain (NBD)1 of the ClpB chaperone induces motion of the long coiled-coil, stabilizes the hexamer, and activates NBD2.
      ,
      • Oguchi Y.
      • Kummer E.
      • Seyffer F.
      • Berynskyy M.
      • Anstett B.
      • Zahn R.
      • Wade R.C.
      • Mogk A.
      • Bukau B.
      A tightly regulated molecular toggle controls AAA+ disaggregase.
      ,
      • Sielaff B.
      • Tsai F.T.
      The M-domain controls Hsp104 protein remodeling activity in an Hsp70/Hsp40-dependent manner.
      ,
      • Miot M.
      • Reidy M.
      • Doyle S.M.
      • Hoskins J.R.
      • Johnston D.M.
      • Genest O.
      • Vitery M.C.
      • Masison D.C.
      • Wickner S.
      Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation.
      ,
      • Rosenzweig R.
      • Moradi S.
      • Zarrine-Afsar A.
      • Glover J.R.
      • Kay L.E.
      Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction.
      ,
      • Seyffer F.
      • Kummer E.
      • Oguchi Y.
      • Winkler J.
      • Kumar M.
      • Zahn R.
      • Sourjik V.
      • Bukau B.
      • Mogk A.
      Hsp70 proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces.
      ).
      The ATPase modules NBD1 and NBD2 are the motors that drive the molecular machine in a cooperative fashion. The catalytic sites, which are located at the interface between two subunits in the hexameric complex, are built up by highly conserved motifs, namely the Walker A and B motifs that are crucial for nucleotide binding and ATP hydrolysis, respectively (
      • Walker J.E.
      • Saraste M.
      • Runswick M.J.
      • Gay N.J.
      Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.
      ,
      • Wendler P.
      • Ciniawsky S.
      • Kock M.
      • Kube S.
      Structure and function of the AAA+ nucleotide binding pocket.
      ). Furthermore, there are essential arginine residues, often termed arginine fingers, that contribute to the active sites in trans because they are in close proximity to the nucleotide bound to the adjacent subunit. The role of such conserved arginines in AAA+ proteins has been investigated extensively (
      • Ogura T.
      • Whiteheart S.W.
      • Wilkinson A.J.
      Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAA+ ATPases.
      ,
      • Yamasaki T.
      • Nakazaki Y.
      • Yoshida M.
      • Watanabe Y.H.
      Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.
      ,
      • Biter A.B.
      • Lee J.
      • Sung N.
      • Tsai F.T.
      • Lee S.
      Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine.
      ,
      • Wang Q.
      • Song C.
      • Irizarry L.
      • Dai R.
      • Zhang X.
      • Li C.C.
      Multifunctional roles of the conserved Arg residues in the second region of homology of p97/valosin-containing protein.
      ). However, it is not trivial to distinguish a truly catalytic arginine finger, as initially identified in GTPase-activating proteins (
      • Ahmadian M.R.
      • Stege P.
      • Scheffzek K.
      • Wittinghofer A.
      Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras.
      ), from conserved arginines that either stabilize the hexameric state or are crucial for allosteric regulation. The complexity is even increased by the fact that several AAA+ proteins, such as ClpB/Hsp104, ClpA, ClpC, and p97/VCP/Cdc48, possess two highly conserved, neighboring arginines in their NBD1 subunit interface. With this study, we aimed at understanding the mechanistic role of this essential, trans-acting pair of arginines in allosteric communication between AAA+ subunits.
      We showed previously that NBD1-M and NBD2 of ClpB from T. thermophilus can be expressed and purified separately (
      • Beinker P.
      • Schlee S.
      • Auvula R.
      • Reinstein J.
      Biochemical coupling of the two nucleotide binding domains of ClpB: covalent linkage is not a prerequisite for chaperone activity.
      ), which allowed a detailed and quantitative characterization of both AAA+ motor domains with regard to nucleotide binding, oligomerization, and activity (
      • Werbeck N.D.
      • Kellner J.N.
      • Barends T.R.M.
      • Reinstein J.
      Nucleotide Binding and Allosteric Modulation of the second AAA+ domain of ClpB probed by transient kinetic studies.
      ,
      • Werbeck N.D.
      • Zeymer C.
      • Kellner J.N.
      • Reinstein J.
      Coupling of oligomerization and nucleotide binding in the AAA+ chaperone ClpB.
      ,
      • Zeymer C.
      • Barends T.R.
      • Werbeck N.D.
      • Schlichting I.
      • Reinstein J.
      Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor.
      ,
      • Zeymer C.
      • Werbeck N.D.
      • Schlichting I.
      • Reinstein J.
      The molecular mechanism of Hsp100 chaperone inhibition by the prion curing agent guanidinium chloride.
      ). Here, we used the construct NBD1-M. Inspired by successful work on the mechanism of ClpX, another AAA+ protein (
      • Martin A.
      • Baker T.A.
      • Sauer R.T.
      Rebuilt AAA+ motors reveal operating principles for ATP-fuelled machines.
      ,
      • Glynn S.E.
      • Martin A.
      • Nager A.R.
      • Baker T.A.
      • Sauer R.T.
      Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine.
      ,
      • Stinson B.M.
      • Nager A.R.
      • Glynn S.E.
      • Schmitz K.R.
      • Baker T.A.
      • Sauer R.T.
      Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine.
      ), we applied a combined approach of covalently linking NBD1-M subunits and introducing Walker A/B and arginine finger mutations. Using these fixed and well determined arrangements of wild-type and mutated subunits in a direct neighborhood, it was possible to dissect the mechanisms of allosteric regulation and intersubunit communication in the AAA+ chaperone ClpB/Hsp104.

      DISCUSSION

      In this study, we investigated the role of the conserved, trans-acting arginines Arg-322 and Arg-323 in allosteric regulation and intersubunit communication in the molecular disaggregation machine ClpB (Fig. 5). Using a simplified system, namely the separate N-terminal ATPase subunit NBD1-M, it was possible to study the interplay between nucleotide binding, oligomerization, and activity in a quantitative manner. We utilized a set of well defined NBD1-M dimers with intermolecular disulfide cross-links and site-specifically introduced Walker A/B mutations to draw conclusions about allosteric effects mediated by the conserved arginine pair.
      Figure thumbnail gr5
      FIGURE 5Allosteric regulation and intersubunit communication in ClpB NBD1. Putative pathways of allosteric regulation (orange arrows) are illustrated schematically. The N-terminal ATPase subunit of ClpB, NBD1, is shown as a gray sphere. ATP is depicted in green and the trans-acting arginines Arg-322 and Arg-323 are shown in blue and pink, respectively. The active site is located at the interface between two subunits in the oligomeric ClpB complex. The conserved arginines interact with the γ-phosphate of the ATP molecule bound to the neighboring subunit, which presumably is the structural basis for intersubunit communication. The nucleotide state of the adjacent active site is sensed and communicated throughout the AAA+ domain, which allosterically regulates the activity of the whole oligomeric complex. This process may involve other highly conserved residues that are part of nucleotide sensor motifs in cis and induce conformational changes throughout the ATPase cycle.
      First, we showed that both arginines are involved in coupling nucleotide binding to oligomerization of ClpB NBD1-M. We identified the NBD1-M trimer as the smallest ATP hydrolysis-competent unit, which is formed upon ATP binding, but only if both arginines are present. The finding that trimer formation is essential is in agreement with previously performed mixing experiments showing that the random incorporation of two mutant ClpB subunits into the hexamer is sufficient to abolish activity (
      • Werbeck N.D.
      • Schlee S.
      • Reinstein J.
      Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB.
      ). A previous study using full-length ClpB came to the conclusion that the trans-acting arginines are not involved in nucleotide binding (
      • Yamasaki T.
      • Nakazaki Y.
      • Yoshida M.
      • Watanabe Y.H.
      Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.
      ). However, using the cross-linked dimers, we showed that the arginines are indeed crucial for strong and cooperative ATP binding.
      The next goal was to obtain a better understanding of allosteric regulation mechanisms implemented in ClpB NBD1-M. A comprehensive mechanistic interpretation of the nucleotide binding, oligomerization, and activity data that we obtained for the different cross-linked dimer variants would greatly benefit from additional structural information about the ClpB subunit interface. The available crystal structure of ClpB from T. thermophilus (Protein Data Bank code 1QVR) exhibits a helical arrangement of subunits, thereby displaying a shifted subunit interface (
      • Lee S.
      • Sowa M.E.
      • Watanabe Y.-H.
      • Sigler P.B.
      • Chiu W.
      • Yoshida M.
      • Tsai F.T.F.
      The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state.
      ). The two conserved arginines Arg-322 and Arg-323 are located 5 and 11 Å away from the γ-phosphate of ATP bound to the neighboring subunit, respectively (see Fig. 2A), which may not reflect the active conformation. Several cryo-EM studies on ClpB and its yeast homolog Hsp104 generated models of a planar hexameric ring, which is believed to be the active form (
      • Lee S.
      • Sowa M.E.
      • Watanabe Y.-H.
      • Sigler P.B.
      • Chiu W.
      • Yoshida M.
      • Tsai F.T.F.
      The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state.
      ,
      • Lee S.
      • Choi J.-M.
      • Tsai F.T.F.
      Visualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB.
      ,
      • Lee S.
      • Sielaff B.
      • Lee J.
      • Tsai F.T.
      CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation.
      ,
      • Carroni M.
      • Kummer E.
      • Oguchi Y.
      • Wendler P.
      • Clare D.K.
      • Sinning I.
      • Kopp J.
      • Mogk A.
      • Bukau B.
      • Saibil H.R.
      Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation.
      ). However, structural details, such as the conformation of the conserved arginines, could not be resolved. When using the hexameric crystal structure of the highly homologous AAA+ protein ClpC together with its adaptor protein MecA (Protein Data Bank code 3PXG) as a template for a planar ClpB model, both conserved arginines are at a 4–6-Å distance from a modeled ATP molecule (
      • Wang F.
      • Mei Z.
      • Qi Y.
      • Yan C.
      • Hu Q.
      • Wang J.
      • Shi Y.
      Structure and mechanism of the hexameric MecA-ClpC molecular machine.
      ). Still, at this point, there is no reliable knowledge about the exact positioning of the conserved arginine pair in the ClpB subunit interface. Thus, we put great emphasis on control experiments using different Walker A/B mutants to verify our results.
      Allosteric effects related to Walker A/B mutations were studied previously, mainly by using mixing experiments (
      • Werbeck N.D.
      • Schlee S.
      • Reinstein J.
      Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB.
      ,
      • Franzmann T.M.
      • Czekalla A.
      • Walter S.G.
      Regulatory circuits of the AAA+ disaggregase Hsp104.
      ,
      • DeSantis M.E.
      • Leung E.H.
      • Sweeny E.A.
      • Jackrel M.E.
      • Cushman-Nick M.
      • Neuhaus-Follini A.
      • Vashist S.
      • Sochor M.A.
      • Knight M.N.
      • Shorter J.
      Operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients.
      ,
      • Fernández-Higuero J.Á.
      • Acebrón S.P.
      • Taneva S.G.
      • Del Castillo U.
      • Moro F.
      • Muga A.
      Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B.
      ). The extensive amount of experimental data was complemented recently by a computational study simulating cooperativity in AAA+ proteins (
      • Le D.T.
      • Eckert T.
      • Woehlke G.
      Computer simulation of assembly and co-operativity of hexameric AAA ATPases.
      ). Franzmann et al. (
      • Franzmann T.M.
      • Czekalla A.
      • Walter S.G.
      Regulatory circuits of the AAA+ disaggregase Hsp104.
      ) analyzed the allosteric network in Hsp104 and found regulatory circuits in both cis and trans. They concluded that the ATPase activity of a given NBD1 depends on the nucleotide state of the neighboring subunit, which our experiments fully agree with. However, in contrast to this previous study, we observed that the presence of a nucleotide binding-deficient subunit (Walker A mutation) inhibits ATP hydrolysis in the neighboring, intact NBD1-M unit, whereas the presence of a tightly bound ATP in a hydrolysis-deficient subunit (Walker B mutation) activates the ATPase activity of the direct neighbor. One could speculate that this regulatory feature ensures a concerted action of several subunits in the oligomeric ClpB complex, which is in agreement with previous work by DeSantis et al. (
      • DeSantis M.E.
      • Leung E.H.
      • Sweeny E.A.
      • Jackrel M.E.
      • Cushman-Nick M.
      • Neuhaus-Follini A.
      • Vashist S.
      • Sochor M.A.
      • Knight M.N.
      • Shorter J.
      Operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients.
      ) showing that ClpB indeed hydrolyzes ATP in a cooperative fashion, whereas ATP hydrolysis in Hsp104 is generally probabilistic unless the substrate is a stable amyloid.
      The role of conserved, trans-acting arginines in AAA+ proteins was analyzed previously (
      • Ogura T.
      • Whiteheart S.W.
      • Wilkinson A.J.
      Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAA+ ATPases.
      ,
      • Yamasaki T.
      • Nakazaki Y.
      • Yoshida M.
      • Watanabe Y.H.
      Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.
      ,
      • Biter A.B.
      • Lee J.
      • Sung N.
      • Tsai F.T.
      • Lee S.
      Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine.
      ,
      • Wang Q.
      • Song C.
      • Irizarry L.
      • Dai R.
      • Zhang X.
      • Li C.C.
      Multifunctional roles of the conserved Arg residues in the second region of homology of p97/valosin-containing protein.
      ). Studies on ClpB and Hsp104 agree that the arginines in NBD1 are crucial for both oligomerization and activity. Notably, using our set of cross-linked ClpB NBD1-M dimers, we could (i) distinguish between regulatory functions and oligomerization effects, (ii) observe the influence of arginine mutations in a well defined environment of site-specifically engineered neighboring subunits, and (iii) be independent from overlaying allosteric effects caused by NBD2. In summary, our data indicate that the conserved arginines not only mediate the coupling between nucleotide binding and oligomerization but indeed regulate ATP hydrolysis in a truly allosteric fashion, namely by influencing a catalytic site that they do not directly interact with. Without these arginines, the cooperativity of ATP binding and hydrolysis is completely lost, even if oligomerization is ensured by chemical linkage.
      It remains an open question why two such arginines are found in NBD1 of several AAA+ proteins, such as ClpB/Hsp104, ClpA, ClpC, and p97/VCP/Cdc48. Wang et al. (
      • Wang Q.
      • Song C.
      • Irizarry L.
      • Dai R.
      • Zhang X.
      • Li C.C.
      Multifunctional roles of the conserved Arg residues in the second region of homology of p97/valosin-containing protein.
      ) studied the function of this conserved arginine pair in the N-terminal AAA+ domain of p97, which is involved in various cellular processes that are directly or indirectly regulated by the ubiquitin-proteasome system. They also concluded that one arginine is more important for maintaining the hexameric state than the other, but both arginines are essential for intersubunit communication and stimulation of the ATPase activity. Clearly, both arginines are essential and cannot replace each other's function. They may have to work in concert to sense and communicate the nucleotide state and thus facilitate fine tuning of the activity in AAA+ protein complexes.

      Acknowledgments

      We thank Sabine Zimmermann, Susanne Eisel, and Melanie Müller for excellent technical assistance and acknowledge Susann Mönchgesang for work during a laboratory rotation.

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