The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients

Abstract

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins. The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins. Hsp70 is an ATP-dependent molecular chaperone that is abundantly expressed in most cancer cells to facilitate the maturation, activation, and stabilization of many oncogenic clients to buffer cellular stress (1Murphy M.E. The HSP70 family and cancer.Carcinogenesis. 2013; 34 (23563090): 1181-118810.1093/carcin/bgt111Crossref PubMed Scopus (379) Google Scholar, 2Sherman M.Y. Gabai V.L. Hsp70 in cancer: back to the future.Oncogene. 2015; 34 (25347739): 4153-416110.1038/onc.2014.349Crossref PubMed Scopus (153) Google Scholar, 3Wu J.M. Liu T.E. Rios Z. Mei Q.B. Lin X.K. Cao S.S. Heat shock proteins and cancer.Trends Pharmacol. Sci. 2017; 38 (28012700): 226-25610.1016/j.tips.2016.11.009Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). The chaperone function of Hsp70 is based on an allosteric mechanism, in which nucleotide-binding and conformation-specific co-chaperones regulate Hsp70-client protein interaction. As such, recruitment of Hsp70 by these co-chaperones would create a local pool of the chaperone to facilitate folding of clients (4Taipale M. Tucker G. Peng J. Krykbaeva I. Lin Z.Y. Larsen B. Choi H. Berger B. Gingras A.C. Lindquist S. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.Cell. 2014; 158 (25036637): 434-44810.1016/j.cell.2014.05.039Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 5Clerico E.M. Tilitsky J.M. Meng W.L. Gierasch L.M. How Hsp70 molecular machines interact with their substrates to mediate diverse physiological functions.J. Mol. Biol. 2015; 427 (25683596): 1575-158810.1016/j.jmb.2015.02.004Crossref PubMed Scopus (208) Google Scholar). Besides, Hsp70 inhibits caspase-independent and caspase-dependent apoptosis by directly interacting with apoptosis-inducing factors, such as AIF and APAF-1, and inhibits caspase 3 and 9 activations (6Creagh E.M. Carmody R.J. Cotter T.G. Heat shock protein 70 inhibits caspase-dependent and -independent apoptosis in Jurkat T cells.Exp. Cell Res. 2000; 257 (10854054): 58-6610.1006/excr.2000.4856Crossref PubMed Scopus (169) Google Scholar, 7Beere H.M. The stress of ding': the role of heat shock proteins in the regulation of apoptosis.J. Cell Sci. 2004; 117 (15169835): 2641-265110.1242/jcs.01284Crossref PubMed Scopus (508) Google Scholar). However, as pivotal regulators of the mitochondrial apoptosis pathway, Bcl-2 family members have cross-talk with Hsp70 in a way that is not fully understood (8Rouf R. Kadry S. Molecular chaperone HSP70 and key regulators of apoptosis: a review.Curr. Mol. Med. 2019; 19 (30914024): 315-32510.2174/1566524019666190326114720Crossref PubMed Scopus (24) Google Scholar). Although a few previous reports have found Bim and Bax interactions with Hsp70 by co-IP and FRET (9Ambrosini G. Seelman S.L. Schwartz G.K. Differentiation-related gene-1 decreases Bim stability by proteasome-mediated degradation.Cancer Res. 2009; 69 (19622774): 6115-612110.1158/0008-5472.CAN-08-3024Crossref PubMed Scopus (14) Google Scholar, 10Gotoh T. Terada K. Oyadomari S. Mori M. Hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria.Cell Death Differ. 2004; 11 (14752510): 390-40210.1038/sj.cdd.4401369Crossref PubMed Scopus (294) Google Scholar, 11Park S.H. Ko W. Park S.H. Lee H.S. Shin I. Evaluation of the interaction between Bax and Hsp70 in cells by using a FRET system consisting of a fluorescent amino acid and YFP as a FRET pair.ChemBiochem. 2020; 21 (31206981): 59-6310.1002/cbic.201900293Crossref PubMed Scopus (6) Google Scholar), there is no solid evidence of the physical interactions between them. The biological consequence of these complexes is unknown. The protein-protein interactions (PPIs) of the Bcl-2 family proteins dictate apoptosis, which is mediated by a Bcl-2 homology 3 (BH3) domain of the pro-apoptotic protein that inserts into a BH3 receptor groove on the surface of anti-apoptotic proteins (12Kelekar A. Thompson C.B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis.Trends Cell Biol. 1998; 8 (9704409): 324-33010.1016/S0962-8924(98)01321-XAbstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Recent pieces of evidence have revealed that BH3-only proteins could engage into BH3-receptor proteins besides the 16 well-known Bcl-2 family proteins (13Szlyk B. Braun C.R. Ljubicic S. Patton E. Bird G.H. Osundiji M.A. Matschinsky F.M. Walensky L.D. Danial N.N. A phospho-BAD BH3 helix activates glucokinase by a mechanism distinct from that of allosteric activators.Nat. Struct. Mol. Biol. 2014; 21 (24317490): 36-4210.1038/nsmb.2717Crossref PubMed Scopus (41) Google Scholar, 14Rodriguez D.A. Zamorano S. Lisbona F. Rojas-Rivera D. Urra H. Cubillos-Ruiz J.R. Armisen R. Henriquez D.R. Cheng E.H. Letek M. Vaisar T. Irrazabal T. Gonzalez-Billault C. Letai A. Pimentel-Muinos F.X. et al.BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α.EMBO J. 2012; 31 (22510886): 2322-233510.1038/emboj.2012.84Crossref PubMed Scopus (92) Google Scholar, 15Cooray S. Bahar M.W. Abrescia N.G.A. Mcvey C.E. Bartlett N.W. Chen R.A.J. Stuart D.I. Grimes J.M. Smith G.L. Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.J. Gen. Virol. 2007; 88 (17485524): 1656-166610.1099/vir.0.82772-0Crossref PubMed Scopus (148) Google Scholar), suggesting the presence of unknown BH3 receptors that have cross-talk with Bcl-2 members via a BH3 groove. In this study, Bim was revealed that binds Hsp70 in vitro and in cells through the BH3 domain (a diagram of Bim sequence is shown in Fig. S1). Bim acts as a positive co-chaperone of Hsp70 because it increased ATPase activity and oncogenic chaperone functions of Hsp70 by stabilizing its ADP-binding conformation. In opposite to its classic role as an activator of intrinsic apoptosis, Bim was revealed that could help Hsp70 to stabilize oncogenic clients AKT and Raf-1. We characterized the binding ability of Hsp70 proteins to BH3 domains of Bid, Bim, Noxa, Bax, and Bak by using in vitro biochemical assays and cell-based assays. Two Hsp70 inhibitors (VER-155008 (16Wen W. Liu W.X. Shao Y.F. Chen L. VER-155008, a small molecule inhibitor of HSP70 with potent anti-cancer activity on lung cancer cell lines.Exp. Biol. Med. 2014; 239 (24676905): 638-64510.1177/1535370214527899Crossref PubMed Scopus (68) Google Scholar) and MKT-077 (17Li X.K. Srinivasan S.R. Connarn J. Ahmad A. Young Z.T. Kabza A.M. Zuiderweg E.R.P. Sun D.X. Gestwicki J.E. Analogues of the allosteric heat shock protein 70 (Hsp70) inhibitor, MKT-077, as anti-cancer agents.ACS Med. Chem. Lett. 2013; 4: 1042-104710.1021/ml400204nCrossref Scopus (110) Google Scholar)) were tested in parallel. First, the specific binding effects of these BH3 peptides on apo-Hsp70 (nucleotide-free Hsp70) were evaluated through FP. As shown in Fig. S2, BH3 peptides derived from Bid, Bim, Noxa, Bax, and Bak exhibited direct binding affinities (Kd = 1.3-1.5 μm) to apo-Hsp70. MKT-077 could effectively compete the binding of BimBH3 to Hsp70 = to the BimBH3 and to it the binding of BimBH3 to Hsp70 in (Kd that the direct interactions of Bcl-2 family proteins with Hsp70 mediated by the BH3 where MKT-077 but not also Hsp70 could Bcl-2 members through the BH3 domain in were in and cells to multidomain pro-apoptotic members and BH3-only pro-apoptotic members with As shown in Fig. A and the binding of Hsp70 were found to in the cell Bim was in with Hsp70 in cell Bax and Bak were found to with Hsp70 in suggesting the cell Hsp70 with Bcl-2 proteins. interactions were found between BH3-only proteins Noxa, and with Hsp70 in of and We further the cell to and and Bim interactions with Hsp70, interactions with BH3-only proteins were not but in and As shown in Fig. A and BimBH3 the that Bim interacts with Hsp70 via BH3 domain in The control was In the was by MKT-077, to no was found for which is with the in vitro binding the of Bim for it was by MKT-077, and it not show with to to protein into clients and the ATPase activity of Hsp70 is by for MKT-077, the protein would A. A. T. M.E. of the activity of HSP70 inhibitors on apoptosis, cell and Biol. 2014; PubMed Scopus Google Scholar). The Bim MKT-077 that Bim is to a co-chaperone of Hsp70 a BimBH3 binds to Hsp70 binding (Kd the the affinities of the proteins to Shao H. Srinivasan S.R. E. C. Zuiderweg E.R.P. D.R. Gestwicki J.E. inhibitor of apoptosis protein is a of heat shock protein 70 (Hsp70) and a of its Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which also that Bim a co-chaperone of Hsp70 proteins a the biological of Bcl-2 family proteins with Hsp70 from a the for and of Bcl-2 family protein with Hsp70 were in the = 16 S1). As shown in Fig. and Fig. there is a between Bim and Hsp70 = = of J. for the L. Scholar). with Bim, there were positive between pro-apoptotic BH3-only and multidomain Bcl-2 family members with The a biological function between Hsp70 and Bim. on in the the interaction site for Bim on Hsp70, expressed the and of Hsp70, and the interaction of Bim with of Hsp70 by isothermal titration As shown in Fig. a binding for Hsp70 (Kd = μm) to Hsp70 (Kd = μm) that exhibited binding and for Hsp70 domain (Kd = μm) to Hsp70 (Kd = μm) and the interactions of the domain to (Kd = μm) Although BimBH3 binds and no binding was in the of to Hsp70, suggesting that a of and in Hsp70 binding for Bim, as as found in the A. K. I. insights into a interaction in the Struct. Mol. Biol. 2017; PubMed Scopus (14) Google Scholar). The binding of Bim to that BimBH3 interacts with Hsp70 in an and by binding site and of Bim in Hsp70 as by and trypsin isothermal titration of the interaction of with Hsp70 Hsp70 and Hsp70 of heat for the titration of μm) into Hsp70 the binding The binding site and were from The were shown as of of with the of BimBH3 in a of the of BimBH3 and in the of and the of Hsp70 BimBH3 The of are as where the of the and the of the of an amino acid = of the with on and show the binding site of MKT-077 and domain, as trypsin of Hsp70 of domain and BimBH3 and were in an and the of these is shown in the are the = The of were evaluated by BimBH3 that Bim, the binding of As shown in Fig. BimBH3 with Hsp70 (Kd = Hsp70 (Kd = and Hsp70 domain (Kd = μm) as as it that Bim interacts with the Hsp70 domain via the BH3 1H-15N-transverse relaxation optimized spectroscopy to the binding site of Bim on Hsp70 that binds BimBH3 as as Hsp70 which is with the in Hsp70 and of the of the domain have in E.R.P. Gestwicki J.E. and of the binding domain in the 2017; 11 PubMed Scopus Google Scholar), a of to the binding site The of and a in and Fig. the in and of the in were in and a opposite to the nucleotide-binding and this is as the binding site of BimBH3 the binding of the most in and the on Bim binding that with Hsp70 the in a of binding the of the interaction site of Bim on with MKT-077 and binding were in and BimBH3 binds into a site distinct from of them. is a in MKT-077 and of is with the of MKT-077 BimBH3 in and co-IP assays. The that BimBH3 in a site that is from the by the binding between BimBH3 and Hsp70 in the presence of the and that the the interaction between BimBH3 and Hsp70 The that the binding site of Bim is from proteins small that on the Hsp70 domain a between the and Bim has such an trypsin of with previous reports L. Zuiderweg E.R.P. Gestwicki J.E. a regulation of through an allosteric Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), the of the Hsp70 with was into including and with of domain, which in Hsp70, the into an as shown by a of that in Fig. of BimBH3 to Hsp70 which is to the of and that Bim binds to the of The identified site is a novel allosteric site by which the of Hsp70 could the solid evidence of Bim binding with Hsp70 to as an allosteric has which to the of BimBH3 on ATPase and chaperone of We evaluated the of Bim on Hsp70 As shown in Fig. the of Hsp70 μm) and BimBH3 which the of Hsp70 in Hsp70 as by in cells in a of the of to the of domain and of their of ATPase activity were by The control was We also BimBH3 with a Hsp70's As shown in Fig. A and BimBH3 could stimulate the ATPase activity of the Hsp70 domain by which is to that of Hsp70 In Hsp70 but not Hsp70 with BimBH3 domain and with BimBH3 domain by and suggesting that the could to stimulate ATPase of However, the is when using the Hsp70 domain suggesting that is for the the aggregation of denatured rhodanese by to the of BimBH3 on Hsp70 As shown in Fig. the of BimBH3 domain in Hsp70 rhodanese aggregation by of the positive of BimBH3 on Hsp70 to and stabilize denatured MKT-077 these positive of Hsp70 with Bim binding by the the of BimBH3 on Hsp70 ATPase and aggregation of denatured rhodanese was by the it not the of domain A and In with BimBH3 positive effects of on Hsp70 activity identified that Bim acts as a positive co-chaperone of the Hsp70 chaperone the in vitro biochemical the positive co-chaperone function of Bim in and cells were with Bim As shown in Fig. Bim the Bim protein by Bim not the and of Hsp70, and Hsp70 from cell of and and ATPase assays. As shown by Fig. Bim to a of the ATPase activity of Hsp70 by in and the positive co-chaperone role of Bim in Hsp70 ATPase in Hsp70 is an cancer chaperone that in the folding of oncogenic proteins. The of Hsp70 by Bim because positive co-chaperones could the of clients and most are proteins Molecular and cancer 2013; PubMed Google Scholar). We evaluated regulation of the on AKT and which are well-established clients of Hsp70 and are proteins that control and of cancer cells Peng H.M. for a role of the chaperone in when proteins and Biol. Med. PubMed Scopus Google Scholar). Bim is in the control of these Bim in of AKT and Raf-1. As shown in Fig. Bim a of AKT and Raf-1 by 38 and and Hsp70 a of AKT and Raf-1 by and However, in cells with Hsp70 Bim on the of AKT and Raf-1 Fig. the is for stabilization of AKT and Raf-1. effects of Hsp70 and Bim were in and the cancer cell that these effects are in cancer In with when the were in that a of Hsp70 Hsp70 has no on the of AKT and and Bim the of Hsp70 In the a role to facilitate the chaperone function of Hsp70 proteins, which that of the is to promote the oncogenic chaperone activity of a dual role of Hsp70's anti-apoptotic function was in and cells that Hsp70 not Bim to cells from apoptosis, but also the on stabilizing oncogenic clients with the help of the positive co-chaperone Bim. is of the most Bcl-2 inhibitors that intrinsic apoptosis by pro-apoptotic Bim from Bcl-2 is the Bcl-2 inhibitor S.L. Chen J. H. S.H. S. S.L. et a potent and inhibitor, activity Med. 2013; 19 PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Although a previous has found that from and were with Bim, cells the to J. B. Letai A. Bax from to inhibiting apoptosis thereby PubMed Scopus Google Scholar). a of Hsp70 in in cells the cell with a of and the of Bim and Hsp70 of and (a of were in parallel. As shown in Fig. for a of Bim from which was not to and the is with increased AKT and Raf-1 binding with Hsp70 and of the AKT and Raf-1 protein most of Bim were from and no was In Hsp70 interactions with Bax and proteins that could from by Hsp70 few interactions with Bax and with the cellular activity of the in apoptosis. In in Hsp70 was found in Bim complexes and no of was no of AKT and Raf-1 with Hsp70 and of AKT and Raf-1 were was as as was and the effects were further As by Letai and J. B. Letai A. Bax from to inhibiting apoptosis thereby PubMed Scopus Google Scholar) and in AKT could Bax and Bax from to inhibiting apoptosis, a of could promote to B. L. The inhibitor the stability of and protein in 2014; PubMed Scopus Google Scholar). revealed that it is the of Hsp70 that AKT and Raf-1 with the help of Bim to et G. J. E. M.E. M. D. L. et mitochondrial and in the of cell 2015; Full Text Full Text PDF PubMed Scopus Google Scholar) that and by revealed that Bim by inhibits by stabilization of on the opposite role of Bim as a BH3 activator to by directly it is to that the dual function of Bim to a of which to and further to the role of Bim the of is the to that Bim binds in a allosteric site of Hsp70 through the BH3 domain and its chaperone Bim with the domain has an and binding with Bim domain to ATPase of Hsp70 (a of the was shown in Fig. of a of of BH3-only which BH3-only proteins with on directly the activity and biological their pro-apoptotic function (12Kelekar A. Thompson C.B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis.Trends Cell Biol. 1998; 8 (9704409): 324-33010.1016/S0962-8924(98)01321-XAbstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, B. Braun C.R. Ljubicic S. Patton E. Bird G.H. Osundiji M.A. Matschinsky F.M. Walensky L.D. Danial N.N. A phospho-BAD BH3 helix activates glucokinase by a mechanism distinct from that of allosteric activators.Nat. Struct. Mol. Biol. 2014; 21 (24317490): 36-4210.1038/nsmb.2717Crossref PubMed Scopus (41) Google Scholar, E. G. BH3-only proteins in apoptosis and an PubMed Scopus Google Scholar). However, the on Bim is As the most BH3-only protein that the Bcl-2-like proteins and acts as an activator of multidomain pro-apoptotic Bim such an opposite role to help Hsp70 to stabilize oncogenic are reports that cancer cells a of Bim which a function R. Liu S.H. A. A. Sun L.M. D. Bim, a up-regulated via factor mechanism, functions as a molecule in Biol. Chem. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the molecular mechanism a and could not cancer the of Bim. an mechanism that with Hsp70 to the Bim between and apoptosis. Hsp70 protein was demonstrated to function as a BH3 receptor to Bcl-2 family members via the BH3 domain for the with previous reports of and complexes by co-IP (9Ambrosini G. Seelman S.L. Schwartz G.K. Differentiation-related gene-1 decreases Bim stability by proteasome-mediated degradation.Cancer Res. 2009; 69 (19622774): 6115-612110.1158/0008-5472.CAN-08-3024Crossref PubMed Scopus (14) Google Scholar, 10Gotoh T. Terada K. Oyadomari S. Mori M. Hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria.Cell Death Differ. 2004; 11 (14752510): 390-40210.1038/sj.cdd.4401369Crossref PubMed Scopus (294) Google Scholar), Hsp70 into Bcl-2-like proteins. the binding site of Bim is family such as Hsp70 and as shown by sequence Hsp70 family members are to as BH3 the role of Bim in cell fate regulation and dual function of Hsp70 were revealed in this the solid physical of as as the biological between as by the further of the biological of in cell and Cell and were from and from were in with and of and and of and were into the with a virus site for of the protein The for Hsp70, Hsp70 and were Hsp70 to were to create an with a proteins with an were in the in to an of and by for protein were in where were as were by and the was by for and Hsp70 proteins were from the using acid the The was through The Hsp70 protein was further by the protein was and and and and domain E. was with a that as part of a cells were in of to an of and of the protein was by of to were for and by and in of and were by and the was by for and the was of The was with and The Hsp70 protein interactions with BimBH3 were characterized using an isothermal titration The cell was with Hsp70 Hsp70 in buffer A 3 and buffer 3 by which Hsp70, Hsp70, Hsp70 Hsp70 were and the was with BH3 peptides BimBH3 binding with the the cell was with Hsp70 in buffer of of 3 of was in a with buffer of the protein as a the control were from the The were to a in The were on an with a μm) in and were for the using of BimBH3 in the The was from a A. Gestwicki J.E. Zuiderweg E.R.P. the interaction of MKT-077 with Hsp70 Mol. Biol. PubMed Scopus Google Scholar). Hsp70 μm) in buffer 8 and was with a buffer domain BimBH3 μm) when with were for The trypsin was to a of and the were for The was with the of of buffer and of and to for 3 were by and with ATPase assays were using A of Hsp70 μm) was in buffer and of this and BimBH3 domain and μm) in in buffer were to a and for with MKT-077 The was by in the were for The was with a for of the of a from an buffer the protein was HSP70 ATPase in Bim and Bim HSP70 was from the cell and to ATPase using The aggregation of denatured rhodanese was based on Z.Y. Thompson C. of a novel protein that interacts with heat shock proteins and chaperone Cell Biol. 19 PubMed Scopus Google Scholar) with rhodanese was denatured in for The denatured rhodanese was to a of in buffer 3 in the presence of Hsp70, domain, BimBH3 with The aggregation of rhodanese was by the of The were for the of proteins to the to the for and the in as a of the for rhodanese are under the and the are in this was the for We and for the help the We of of for to the with heat shock protein protein-protein interactions Bcl-2 homology 3 isothermal titration 1H-15N-transverse relaxation optimized spectroscopy fluorescent polarization nucleotide-binding domain

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