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• W2897574976 abstract "ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401–Arg-532 and the inter-subunit pair Asp-404–Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401–Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180–Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities. ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401–Arg-532 and the inter-subunit pair Asp-404–Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401–Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180–Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities. ClpB/Hsp104 is a protein disaggregase required for thermotolerance of cells (1Sanchez Y. Lindquist S.L. HSP104 required for induced thermotolerance.Science. 1990; 248 (2188365): 1112-111510.1126/science.2188365Crossref PubMed Scopus (656) Google Scholar, 2Thomas J.G. Baneyx F. Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG in vivo.J. Bacteriol. 1998; 180 (9748451): 5165-5172Crossref PubMed Google Scholar, 3Weibezahn 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.Cell. 2004; 119 (15550247): 653-66510.1016/j.cell.2004.11.027Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). To disentangle aggregated proteins, ClpB/Hsp104 cooperates with DnaK/Hsp70 chaperone and its co-factors, DnaJ/Hsp40, and the nucleotide exchange factor, GrpE (4Glover J.R. Lindquist S. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins.Cell. 1998; 94 (9674429): 73-8210.1016/S0092-8674(00)81223-4Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 5Motohashi K. Watanabe Y. Yohda M. Yoshida M. Heat-inactivated proteins are rescued by the DnaK.J-GrpE set and ClpB chaperones.Proc. Natl. Acad. Sci. U.S.A. 1999; 96 (10377389): 7184-718910.1073/pnas.96.13.7184Crossref PubMed Scopus (225) Google Scholar, 6Goloubinoff P. Mogk A. Zvi A.P. Tomoyasu T. Bukau B. Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network.Proc. Natl. Acad. Sci. U.S.A. 1999; 96 (10570141): 13732-1373710.1073/pnas.96.24.13732Crossref PubMed Scopus (502) Google Scholar, 7Zolkiewski M. ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli.J. Biol. Chem. 1999; 274 (10497158): 28083-2808610.1074/jbc.274.40.28083Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). As a member of an expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB/Hsp104 contains two AAA+ modules, AAA1 and AAA2, in a polypeptide and forms a hexameric ring-like structure, in which each AAA+ module constitutes each layer of a two layered ring (8Lee S. Sowa M.E. Watanabe Y.H. Sigler P.B. Chiu W. Yoshida M. Tsai F.T. The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state.Cell. 2003; 115 (14567920): 229-24010.1016/S0092-8674(03)00807-9Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). At the top of the ring, highly mobile globular N-terminal domains (ND) 3The abbreviations used are: NDN-terminal domainMDmiddle domainBPMbenzophenone-4-maleimideNBDnucleotide-binding domainSBDsubstrate-binding domainPDBProtein Data BankTCEPtris(2-carboxyethyl)phosphine. contribute to bind protein aggregates (9Barnett M.E. Nagy M. Kedzierska S. Zolkiewski M. The amino-terminal domain of ClpB supports binding to strongly aggregated proteins.J. Biol. Chem. 2005; 280 (16076845): 34940-3494510.1074/jbc.M505653200Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Mizuno S. Nakazaki Y. Yoshida M. Watanabe Y.H. Orientation of the amino-terminal domain of ClpB affects the disaggregation of the protein.FEBS J. 2012; 279 (22348341): 1474-148410.1111/j.1742-4658.2012.08540.xCrossref PubMed Scopus (25) Google Scholar), and long coiled-coil middle domains (MD) protruding from the C-terminal small subdomain of the AAA1 modules surround the main body of the ring (Fig. 1a) (8Lee S. Sowa M.E. Watanabe Y.H. Sigler P.B. Chiu W. Yoshida M. Tsai F.T. The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state.Cell. 2003; 115 (14567920): 229-24010.1016/S0092-8674(03)00807-9Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). The MD is subdivided into two oppositely-directed short coiled-coils, motif-1 and motif-2. N-terminal domain middle domain benzophenone-4-maleimide nucleotide-binding domain substrate-binding domain Protein Data Bank tris(2-carboxyethyl)phosphine. Both the AAA1 and AAA2 modules bind and hydrolyze ATP in a complex cooperative manner that contains intra- and inter-ring cooperation (11Fernández-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.J. Biol. Chem. 2011; 286 (21642426): 25547-2555510.1074/jbc.M111.231365Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 12Franzmann T.M. Czekalla A. Walter S.G. Regulatory circuits of the AAA+ disaggregase Hsp104.J. Biol. Chem. 2011; 286 (21454552): 17992-1800110.1074/jbc.M110.216176Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13Yamasaki T. Oohata Y. Nakamura T. Watanabe Y.H. Analysis of the cooperative ATPase cycle of the AAA+ chaperone ClpB from Thermus thermophilus by using ordered heterohexamers with an alternating subunit arrangement.J. Biol. Chem. 2015; 290 (25713084): 9789-980010.1074/jbc.M114.617696Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The structural changes of the ring, induced by the cooperative ATPase cycle, promote disentangling of aggregated substrates by threading them into the central pore of the ring. Recent cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy analyses demonstrated that the structural inter-conversion between the hexameric ring, spiral, and twisted half-spiral structures occurred during the ATPase cycle and contributed to the disaggregation reaction (14Yokom A.L. Gates S.N. Jackrel M.E. Mack K.L. Su M. Shorter J. Southworth D.R. Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation.Nat. Struct. Mol. Biol. 2016; 23 (27478928): 830-83710.1038/nsmb.3277Crossref PubMed Scopus (82) Google Scholar, 15Gates S.N. Yokom A.L. Lin J. Jackrel M.E. Rizo A.N. Kendsersky N.M. Buell C.E. Sweeny E.A. Mack K.L. Chuang E. Torrente M.P. Su M. Shorter J. Southworth D.R. Ratchet-like polypeptide translocation mechanism of the AAA+ disaggregase Hsp104.Science. 2017; 357 (28619716): 273-27910.1126/science.aan1052Crossref PubMed Scopus (174) Google Scholar, 16Deville C. Carroni M. Franke K.B. Topf M. Bukau B. Mogk A. Saibil H.R. Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.Sci. Adv. 2017; 3 (28798962): e170172610.1126/sciadv.1701726Crossref PubMed Scopus (83) Google Scholar, 17Uchihashi T. Watanabe Y.H. Nakazaki Y. Yamasaki T. Watanabe H. Maruno T. Ishii K. Uchiyama S. Song C. Murata K. Iino R. Ando T. Dynamic structural states of ClpB involved in its disaggregation function.Nat. Commun. 2018; 9 (29858573): 214710.1038/s41467-018-04587-wCrossref PubMed Scopus (40) Google Scholar). Ordinarily, the MD represses the ATPase activity of ClpB/Hsp104 by an unknown mechanism (18Oguchi 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.Nat. Struct. Mol. Biol. 2012; 19 (23160353): 1338-134610.1038/nsmb.2441Crossref PubMed Scopus (101) Google Scholar, 19Lipińska N. Zíe¸tkiewicz S. Sobczak A. Jurczyk A. Potocki W. Morawiec E. Wawrzycka A. Gumowski K. Ślusarz M. Rodziewicz-Motowid;̸o S. Chruściel E. Liberek K. Disruption of ionic interactions between the nucleotide binding domain 1 (NBD1) and middle (M) domain in Hsp100 disaggregase unleashes toxic hyperactivity and partial independence from Hsp70.J. Biol. Chem. 2013; 288 (23233670): 2857-286910.1074/jbc.M112.387589Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In addition, the binding affinity of ClpB/Hsp104 for aggregation is not high enough to start threading (20Acebrón S.P. Martín I. del Castillo U. Moro F. Muga A. DnaK-mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface.FEBS Lett. 2009; 583 (19698713): 2991-299610.1016/j.febslet.2009.08.020Crossref PubMed Scopus (58) Google Scholar). To perform disaggregation, the recruitment of ClpB/Hsp104 to the aggregates and the cancellation of repression are required (21Hayashi S. Nakazaki Y. Kagii K. Imamura H. Watanabe Y.H. Fusion protein analysis reveals the precise regulation between Hsp70 and Hsp100 during protein disaggregation.Sci. Rep. 2017; 7 (28819163): 864810.1038/s41598-017-08917-8Crossref PubMed Scopus (10) Google Scholar). DnaK/Hsp70 consists of an N-terminal nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) (22Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein.Nature. 1990; 346 (2143562): 623-62810.1038/346623a0Crossref PubMed Scopus (830) Google Scholar, 23Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Structural analysis of substrate binding by the molecular chaperone DnaK.Science. 1996; 272 (8658133): 1606-161410.1126/science.272.5268.1606Crossref PubMed Scopus (1060) Google Scholar). The NBD controls the conformation of the SBD and its binding affinity for substrate proteins in an ATPase cycle-dependent manner (24Kityk R. Kopp J. Sinning I. Mayer M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.Mol. Cell. 2012; 48 (23123194): 863-87410.1016/j.molcel.2012.09.023Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 25Zhuravleva A. Clerico E.M. Gierasch L.M. An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones.Cell. 2012; 151 (23217711): 1296-130710.1016/j.cell.2012.11.002Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In the high-affinity conformation, the NBD can bind to the edge of MD motif-2 of ClpB/Hsp104 (21Hayashi S. Nakazaki Y. Kagii K. Imamura H. Watanabe Y.H. Fusion protein analysis reveals the precise regulation between Hsp70 and Hsp100 during protein disaggregation.Sci. Rep. 2017; 7 (28819163): 864810.1038/s41598-017-08917-8Crossref PubMed Scopus (10) Google Scholar, 26Rosenzweig R. Moradi S. Zarrine-Afsar A. Glover J.R. Kay L.E. Unraveling the mechanism of protein disaggregation through a ClpB–DnaK interaction.Science. 2013; 339 (23393091): 1080-108310.1126/science.1233066Crossref PubMed Scopus (201) Google Scholar). This interaction causes recruitment of ClpB/Hsp104 to aggregates and relieves repression by the MD. Mutations at the edge of motif-1 and motif-2, E432A and Y503D of Escherichia coli ClpB (EClpB), are known to stabilize the repressed and activated conformations, and these mutants are termed repressed and hyperactive mutants, respectively (18Oguchi 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.Nat. Struct. Mol. Biol. 2012; 19 (23160353): 1338-134610.1038/nsmb.2441Crossref PubMed Scopus (101) Google Scholar, 27Haslberger T. Weibezahn J. Zahn R. Lee S. Tsai F.T. Bukau B. Mogk A. M domains couple the ClpB threading motor with the DnaK chaperone activity.Mol. Cell. 2007; 25 (17244532): 247-26010.1016/j.molcel.2006.11.008Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Single particle reconstitution of cryo-EM images of these mutants revealed that the conformations of the MD in the repressed and hyperactive mutants are horizontal and tilted to the main body, respectively (28Carroni 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.Elife. 2014; 3 (24843029): e0248110.7554/eLife.02481Crossref PubMed Scopus (101) Google Scholar). In the horizontal conformation, both edges of the MD interact with the MD edges of neighboring subunits in a head-to-tail manner. The interaction between the DnaK NBD and the motif-2 of ClpB MD is thought to disrupt the inter-MD interactions and induce the MD into the tilted conformation. Although the role of MD motif-2 in binding DnaK NBD is clear, the roles of motif-1 should also be investigated to understand the mechanisms involved in controlling MD conformation, and in the regulation of ClpB activity by the MD. To clarify these issues, here we identified functionally important residues in motif-1 and their targets for interaction. We also identified the nucleotide dependence of the interactions. To identify important residues in the MD motif-1, we substituted the conserved charged residues, Glu-401, Glu-402, Asp-404, Glu-407, Arg-408, Glu-416, Lys-422, Glu-423, Asp-425, Arg-431, and Glu-438, in motif-1 with Ala and tested the effects on ClpB activities (Fig. 1, b and c). At 55 °C, WT TClpB hydrolyzed ATP at a rate of 48 min−1, and the rate was stimulated by the presence of κ-casein up to 83 min−1 (Fig. 2a). All of the tested Ala mutants showed similar or at most 2-fold higher ATPase activity to that of the WT, in both the absence and presence of κ-casein (Fig. 2a). The disaggregation activities were measured by using α-glucosidase from Bacillus stearothermophilus, which was completely aggregated by heat treatment at 73 °C for 10 min (29Watanabe Y.H. Yoshida M. Trigonal DnaK–DnaJ complex versus free DnaK and DnaJ: heat stress converts the former to the latter, and only the latter can do disaggregation in cooperation with ClpB.J. Biol. Chem. 2004; 279 (14729677): 15723-1572710.1074/jbc.M308782200Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The WT TClpB could reactivate 54% of the aggregated α-glucosidase by incubation with TDnaK, TDnaJ, TGrpE (TKJE), and ATP at 55 °C for 90 min (Fig. 2b). Although the E402A, E407A, E416A, K422A, R431A, and E438A mutants showed almost the same disaggregation activity as that of WT, the reactivation yields of E401A, R408A, and D425A mutants decreased to 10, 24, and 42%, respectively, and the D404A and E423A mutants completely lost disaggregation activity. When the α-glucosidase was heat-treated in the presence of TKJE and ATP, the substrate protein lost enzyme activity (denatured); however, it remained soluble (29Watanabe Y.H. Yoshida M. Trigonal DnaK–DnaJ complex versus free DnaK and DnaJ: heat stress converts the former to the latter, and only the latter can do disaggregation in cooperation with ClpB.J. Biol. Chem. 2004; 279 (14729677): 15723-1572710.1074/jbc.M308782200Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The soluble, denatured protein can be reactivated by incubation at 55 °C in the presence of TKJE, ATP, and TClpB supplemented after the 73 °C heat treatment. In this case, WT TClpB could reactivate 80% of soluble, denatured α-glucosidase (Fig. 2c). As in the case of disaggregation, D404A and E423A mutations severely inhibited the reactivation activity. However, R408A and D425A mutants could reactivate the soluble, denatured α-glucosidase at almost the same efficiency as that of the WT, and the reactivation yield by the E401A mutant also increased, reaching 54%. Here, we identified several important (Glu-401, Arg-408, and Asp-425) and essential (Asp-404 and Glu-423) residues for the chaperone activity of ClpB. Among these residues, the importance of Glu-423 (corresponding to Glu-432 of EClpB) has been reported, and its roles have been proposed previously (18Oguchi 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.Nat. Struct. Mol. Biol. 2012; 19 (23160353): 1338-134610.1038/nsmb.2441Crossref PubMed Scopus (101) Google Scholar, 28Carroni 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.Elife. 2014; 3 (24843029): e0248110.7554/eLife.02481Crossref PubMed Scopus (101) Google Scholar). Thus, hereafter, we focused on Asp-404 and Glu-401. The former is a newly identified essential residue, and the latter is an amino acid that is more important than Arg-408 and Asp-425. To identify the interaction targets of Glu-401 and Asp-404, we used the bidirectional photoreactive cross-linker, benzophenone-4-maleimide (BPM). The BPM-labeled TClpB mutants, E401C-BPM and D404C-BPM, were UV-irradiated in the absence or presence of nucleotides and analyzed by SDS-PAGE (Fig. 3, a and b). By the UV irradiation, the intensity of the band representing noncross-linked BPM-labeled TClpB decreased, and several ladder bands with higher molecular weights appeared (Fig. 3a). As the reaction mixture did not contain proteins other than BPM-labeled TClpB, inter-subunit cross-linking would occur, and each ladder band corresponded to a multimer of BPM-labeled TClpB. We quantified the intensities of bands corresponding to 1–5- and over 6-mer (Fig. 3b). It should be noted that the over 6-mer bands consisted of a few closely positioned bands, which would correspond to linear 6-mer, closed-ring 6-mer, or 7-mer that would appear transiently (17Uchihashi T. Watanabe Y.H. Nakazaki Y. Yamasaki T. Watanabe H. Maruno T. Ishii K. Uchiyama S. Song C. Murata K. Iino R. Ando T. Dynamic structural states of ClpB involved in its disaggregation function.Nat. Commun. 2018; 9 (29858573): 214710.1038/s41467-018-04587-wCrossref PubMed Scopus (40) Google Scholar). When the E401C-BPM was UV-irradiated in the absence of nucleotide, the intensity of the noncross-linked 1-mer band decreased to 24%, and 31% of E401C-BPM appeared as over 6-mer band. However, intensities of the over 6-mer bands decreased to less than 4% when the UV irradiation was performed in the presence of ATP or ADP (Fig. 3, a and b). Also, in the case of D404C-BPM, similar ladder bands appeared after UV irradiation (Fig. 3, a and b). However, the nucleotide dependence was opposite that seen in the case of E401C-BPM. These results indicated that Glu-401 and Asp-404 interacted with neighboring subunits, in different ways, and these interactions were influenced by the nucleotide states. To identify the responsible nucleotide-binding domain, similar experiments using E401C-BPM and D404C-BPM bearing mutations in the Walker-A consensus sequences of AAA1 or AAA2 were also performed (Fig. 3, a and b). Conserved Lys–Thr pairs in the Walker-A sequence (GXXGXGKT) were replaced by Ala–Ala, and the mutated AAA module could not bind nucleotides (30Watanabe Y.H. Motohashi K. Yoshida M. Roles of the two ATP binding sites of ClpB from Thermus thermophilus.J. Biol. Chem. 2002; 277 (11741950): 5804-580910.1074/jbc.M109349200Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In both cases of E401C-BPM and D404C-BPM, Walker-A mutations involving AAA1 were more effective than those in AAA2 in canceling the nucleotide dependence of cross-linking efficiency (Fig. 3, a and b). To identify the site of cross-linkage, we performed mass spectrometric analysis of cross-linked TClpBs by using LC-MS/MS (Table 1). To simplify the interpretation of MS data, ND-truncated TClpB (TClpB_ΔN) was used in this analysis. The TClpB_ΔN_E401C-BPM, UV-irradiated in the absence of nucleotides, was cleaved by trypsin and analyzed by LC-MS/MS. We detected some peptides cross-linked by BPM. Peptides comprising the sequence from Gly-218 to Lys-225 (218GDVPEGLK225) of TClpB were most frequently detected as the cross-linkage targets of E401C-BPM. The peptides 519LEVTEEDIAEIVSRWTGIPVSK540 and 381AIDLIDEAAARLR393 were also detected. When the UV irradiation was performed in the presence of ATP, a similar set of peptides was detected. In this condition, the peptides 519LEVTEEDIAEIVSR532 (a part of 519LEVTEEDIAEIVSRWTGIPVSK540) and 392LRMALESAPCEIDALERKK410 were also detected. In the cases of TClpB_ΔN_D404C-BPM, UV-irradiated in the absence or presence of ATP, peptides containing the sequence 218GDVPEGLK225, 381AIDLIDEAAARLR393, 409KKLQLEIER417, and 701NTVIILTSNLGSPLILEGLQK721 were detected as the cross-linkage targets. A peptide 835VQVDVGPAGLVFAVPAR851 was detected only when the UV irradiation was performed in the presence of ATP. It should be noted that the peptide, 701NTVIILTSNLGSPLILEGLQK721 might be mis-annotated, because the peptide was also detected in the analysis without UV irradiation.Table 1Cross-linked peptide pairs detected by LC-MS/MSTClpBNucleotidePeptide including Cys labeled with BPMPeptide including photo cross-linked residueTheoretical peptide massNo. of detected spectraDaE401CNo nucleotide394MALESAPCEIDALER408218GDVPEGLK2252756.2792412394MALESAPCEIDALER408215IVKGDVPEGLK2253096.5266610394MALESAPCEIDALERK409215IVKGDVPEGLK2253224.621627392LRMALESAPCEIDALERK409218GDVPEGLK2253153.559355394MALESAPCEIDALER408519LEVTEEDIAEIVSRWTGIPVSK5404413.151458394MALESAPCEIDALER408381AIDLIDEAAARLR3933368.649955ATP394MALESAPCEIDALER408218GDVPEGLK2252756.279247394MALESAPCEIDALER408215IVKGDVPEGLK2253096.5266611394MALESAPCEIDALERK409218GDVPEGLK2252884.374192394MALESAPCEIDALER408519LEVTEEDIAEIVSR5323544.670802394MALESAPCEIDALER408519LEVTEEDIAEIVSRWTGIPVSK5404413.1514512394MALESAPCEIDALERK409519LEVTEEDIAEIVSRWTGIPVSK5404541.246402394MALESAPCEIDALER408381AIDLIDEAAARLR3933368.649954394MALESAPCEIDALER408392LRMALESAPCEIDALERKK4104114.995432D404CNo nucleotide394MALESAPEEICALER408218GDVPEGLK2252770.294892394MALESAPEEICALER408215IVKGDVPEGLK2253110.542315394MALESAPEEICALERKK410215IVKGDVPEGLK2253366.732222394MALESAPEEICALER408381AIDLIDEAAARLR3933382.6656011394MALESAPEEICALER408409KKLQLEIER4173112.569192394MALESAPEEICALER408701NTVIILTSNLGSPLILEGLQK721aMALESAPEEICALER-NTVIILTSNLGSPLILEGLQK cross-linked signals were also detected in no UV-irradiation samples.4179.160164ATP394MALESAPEEICALER408215IVKGDVPEGLK2253110.542316394MALESAPEEICALERK409218GDVPEGLK2252898.389842394MALESAPEEICALER408381AIDLIDEAAARLR3933382.665607394MALESAPEEICALER408409KKLQLEIER4173112.569192394MALESAPEEICALER408835VQVDVGPAGLVFAVPAR8513650.823152394MALESAPEEICALER408701NTVIILTSNLGSPLILEGLQK721aMALESAPEEICALER-NTVIILTSNLGSPLILEGLQK cross-linked signals were also detected in no UV-irradiation samples.4179.160164a MALESAPEEICALER-NTVIILTSNLGSPLILEGLQK cross-linked signals were also detected in no UV-irradiation samples. Open table in a new tab To confirm the plausibility of the mass spectrometric analysis, we constructed hexameric structural models of TClpB by homology modeling using recently solved cryo-EM structures of EClpB hexamers as templates (16Deville C. Carroni M. Franke K.B. Topf M. Bukau B. Mogk A. Saibil H.R. Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.Sci. Adv. 2017; 3 (28798962): e170172610.1126/sciadv.1701726Crossref PubMed Scopus (83) Google Scholar). Fig. 4 shows interfaces between an MD motif-1 and the AAA1 of the neighboring subunit in the TClpB models. The MDs in Fig. 4, a and b, correspond to the tilted (chain D of the PDB code 5og1) and the horizontal (chain C of the PDB code 5ofo) conformation, respectively. Although inter-subunit interactions involving Glu-401 and Asp-404 were not identified in these models, the B3-helix (Asp-176–Leu-187) and the loop between the B4-helix and b3-strand (Gly-218–Lys-227) of the AAA1 large sub-domain of the neighboring subunit were found to be proximal to Glu-401 and Asp-404. This was consistent with the result that the 218GDVPEGLK225 peptide was found as the primary cross-linking target of both E401C-BPM and D404C-BPM. On the surface of the helix and the loop, some conserved charged residues, Asp-176, Glu-177, Arg-180, Arg-181, Arg-214, Asp-219, and Glu-222, were found (Fig. 4, a–c). In addition, only in the tilted conformation Glu-401 was located near Arg-532 in the 519LEVTEEDIAEIVSRWTGIPVSK540 peptide that was detected as a cross-linking target for E401C-BPM (Fig. 4, d and e). Furthermore, Arg-532 was highly conserved among various ClpB/Hsp104s (Fig. 4f). Considering the hexameric model, structural regions corresponding to the other detected peptides, 381AIDLIDEAAARLR393, 392LRMALESAPCEIDALERKK410, 409KKLQLEIER417, 835VQVDVGPAGLVFAVPAR851, did not seem to interact with either Glu-401 or Asp-404. Thus, we considered these conserved charged eight residues as candidates for target residues that functionally interact with Glu-401 and/or Asp-404. We substituted these candidate residues with Ala and tested the effects on ClpB activities. D176A and E177A mutants showed 2–4 times higher ATPase activity than the WT, whereas the activities of the other mutants were similar to or slightly lower than the WT (Fig. 5a). D176A, E177A, and E222A mutants could reactivate aggregated and soluble, denatured α-glucosidase with efficiencies comparable with that of WT (Fig. 5, b and c). However, the other mutants hardly reactivated the aggregated α-glucosidase. The reactivation yields of soluble, denatured α-glucosidase by R180A, R181A, R214A, and D219A mutants were also relatively low, ∼20–30%, whereas that of R532A was considerably high at 61%, similar to the case of E401A (Fig. 5, b and c). Because the effects of the R180A, R181A, R214A, D219A, and R532A mutations were similar to those of the E401A and D404A mutations, these residues might interact with Glu-401 and/or Asp-404. To test this possibility, we prepared TClpB mutants in which these charged residues were substituted by amino acids having the opposite charge, and the mutations were combined. All of the single and combined charge-inverted mutants tested here showed comparable ATPase activities to that of the WT (48–150%) and 1.5–2.7-fold ATPase stimulation by κ-casein (Fig. 6, a and b). Although all the single charge-inverted mutants hardly reactivated the heat-aggregated α-glucosidase (the reactivation yields were less than 7%), the R180D_D404R and R532E_E401R double mutants could reactivate it to 31 and 25%, respectively (Fig. 6, c and d). Such recovery of disaggregation activity was not observed with any other combinations of charge-inverted mutations. Concerning the reactivation of soluble, denatured α-glucosidase, some single and combined charge-inverted mutants resulted in some extent of reactivation: R181E (34%), R181D (25%), R214E (50%), R214D (46%), R532E (41%), E401R (20%), and R214D_D404R (27%) (Fig. 6, e and" @default.
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• W2897574976 title "Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation" @default.
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