HspBP1, an Hsp70 Cochaperone, Has Two Structural Domains and Is Capable of Altering the Conformation of the Hsp70 ATPase Domain
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m301109200
ISSN1083-351X
AutoresCatherine A. McLellan, Deborah A. Raynes, Vince Guerriero,
Tópico(s)Heat shock proteins research
ResumoWe present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1–83, is largely unstructured. Domain II, amino acids 84–359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding. We present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1–83, is largely unstructured. Domain II, amino acids 84–359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding. The Hsp70 family of proteins are key components for the response of the cell to stress, as well as for housekeeping functions such as protein folding and signal transduction (1Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2765) Google Scholar, 2Feldman D.E. Frydman J. Curr. Opin. Struct. Biol. 2000; 10: 26-33Crossref PubMed Scopus (166) Google Scholar, 3Nollen E.A.A. Morimoto R.I. J. Cell Sci. 2002; 115: 2809-2816Crossref PubMed Google Scholar). The proteins of this highly conserved family have two structural domains, an amino-terminal ATPase domain and a carboxyl-terminal substrate-binding domain. The two domains of Hsp70 proteins work in a coordinated fashion. The binding and hydrolysis of ATP by the ATPase domain affects the ability of the substrate-binding domain to bind to short hydrophobic sequences on client proteins. Similarly the binding of substrate by the substrate-binding domain stimulates the ATPase activity of the ATP binding domain. The ATPase domain is composed of two large subdomains of approximately equal size, designated I and II. Nucleotides are bound at the base of the large cleft between the subdomains. Each subdomain is further divided into two additional subdomains, designated A and B (4Flaherty K.M. Deluca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar, 5Mayer M.P. Brehmer D. Gassler C.S. Bukau B. Horwich A. Protein Folding in the Cell. Advances in Protein Chemistry. Academic Press, San Diego, CA2002: 1-44Google Scholar).To adapt this mechanism of action to a specific function or cellular milieu, a number of accessory proteins modulate Hsp70 activity. The DnaJ protein family acts in a catalytic fashion to facilitate ATP hydrolysis coordinated to substrate binding. Another category of accessory proteins is nucleotide exchange factors. In prokaryotes, GrpE promotes the dissociation of ADP and the rebinding of ATP to DnaK (the prokaryotic homologue of Hsp70) (5Mayer M.P. Brehmer D. Gassler C.S. Bukau B. Horwich A. Protein Folding in the Cell. Advances in Protein Chemistry. Academic Press, San Diego, CA2002: 1-44Google Scholar). In eukaryotic cytosol the most extensively studied nucleotide exchange factor is Bag1. Comparison of cocrystals of GrpE bound to the DnaK ATPase domain with cocrystals of the Bag domain bound to the Hsc70 ATPase domain shows that, although GrpE and Bag1 have no structural similarity, they induce the same conformational switch in their Hsp70 family partner to promote nucleotide exchange (6Sondermann H. Scheufler C. Schneider C. Hohfeld J. Hartl F.-U. Moarefi I. Science. 2001; 291: 1553-1557Crossref PubMed Scopus (360) Google Scholar). These nucleotide exchange factors promote an open conformation of the ATPase domain, which is characterized by a 14o rotation of subdomain IIB outward along with a reorientation of the amino terminus in subdomain IA. These two regions are thought to be critical for orienting the nucleotide moiety and act together like a clamp.HspBP1 is a nucleotide exchange factor found in mammalian cytosol. HspBP1 has been shown to bind to the ATPase domain of Hsp70 and inhibit its ATPase activity as well as its ability to renature luciferase (7Raynes D.A. Guerriero V. J. Biol. Chem. 1998; 273: 32883-32888Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Recently HspBP1 has also been shown to promote nucleotide dissociation from Hsc70 (8Kabani M. McLellan C. Raynes D.A. Guerriero V. Brodsky J.L. FEBS Lett. 2002; PubMed Google Scholar). There is growing evidence that HspBP1 is a member of a new class of nucleotide exchange factors. HspBP1 has been shown to be a homologue of the yeast nucleotide exchange factors fes1p and sls1p that are found in the cytosol and endoplasmic reticulum, respectively (8Kabani M. McLellan C. Raynes D.A. Guerriero V. Brodsky J.L. FEBS Lett. 2002; PubMed Google Scholar, 9Kabani M. Beckerich J.M. Gaillardin C. Mol. Cell. Biol. 2000; 277: 6923-6934Crossref Scopus (69) Google Scholar, 10Kabani M. Beckerich J.M. Brodsky J.L. Mol. Cell. Biol. 2002; : 4677-4689Crossref PubMed Scopus (114) Google Scholar). HspBP1 also has significant similarity to Sil1p, the human homologue of yeast sls1p (11Tyson J.R. Stirling C.J. EMBO J. 2000; 19: 6440-6452Crossref PubMed Google Scholar). BAP, a protein found in mammalian endoplasmic reticulum, also acts as a nucleotide exchange factor and shares some sequence homology with HspBP1 (10Kabani M. Beckerich J.M. Brodsky J.L. Mol. Cell. Biol. 2002; : 4677-4689Crossref PubMed Scopus (114) Google Scholar, 12Chung K.T. Shen Y. Hendershot L.M. J. Biol. Chem. 2002; : 47557-47563Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). There has been no structural information reported for HspBP1, though it has been predicted to have two regions that have similarity to armadillo repeats (12Chung K.T. Shen Y. Hendershot L.M. J. Biol. Chem. 2002; : 47557-47563Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar).In this study, using limited proteolysis, we define two structural domains of HspBP1. We also show that interaction of HspBP1 or domain II (the proteolytically stable domain of HspBP1) with the Hsp70 ATPase domain is capable of changing the conformation of the ATPase domain. We further evaluate truncation mutants of HspBP1 for their ability to bind to Hsp70 and to inhibit luciferase renaturation. Domain II is sufficient for both these functions, but no mutant tested was as potent as full-length HspBP1.EXPERIMENTAL PROCEDURESConstruction of Mutants—To construct the HspBP1 mutants, new stop and start sites were engineered using PCR mutagenesis. One mutagenic primer (to add the ATG or stop codon) and an internal primer were used. The PCR product was then subcloned into pCR2.1 (Invitrogen) and sequenced (Genomic Analysis Technology Core, University of Arizona). This cassette was then inserted into the pET28a-HspBP1 full-length vector (7Raynes D.A. Guerriero V. J. Biol. Chem. 1998; 273: 32883-32888Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) using NdeI and a unique internal restriction site spanned by the PCR fragment to create a new start site or an internal restriction site and EcoRI (from pCR2.1 vector) to create a stop.To construct the vector for the Hsp70 ATPase domain, the full-length human Hsp70 clone, pH 2.3 (kindly provided by R. I. Morimoto) (13Hunt C. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6455-6459Crossref PubMed Scopus (693) Google Scholar), was subcloned into pBS+ (Stratagene Corp., La Jolla, CA) and subjected to site-directed mutagenesis to remove an internal NcoI site and create a new NcoI site at the initiation codon. It was then shuttled through pBR328 and subcloned into pET3d (a gift from William Studier). This construct, pET3d-Hsp70, was then subjected to PCR using the T7 primer from the vector and a mutagenic primer to add a stop codon following amino acid 382. The resulting PCR fragment was subcloned into pCR2.1 and sequenced. After the sequence was verified, the fragment was subcloned into pET28a (Novagen, Inc., Madison, WI) with NcoI and EcoRI to remove the His6 tag from the vector.Expression and Purification of HspBP1 Mutants and the Hsp70 ATPase Domain—Recombinant HspBP1 and the HspBP1 truncation mutants all contain amino-terminal His6 tags. These proteins were expressed and purified as previously described for HspBP1 (7Raynes D.A. Guerriero V. J. Biol. Chem. 1998; 273: 32883-32888Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Trace amounts of residual DnaK were removed from the purified HspBP1 and the truncation mutants, using an ATP-agarose column or by washing the TALON (BD Biosciences Clontech, Palo Alto, CA) resin-bound HspBP1 with 25 ml of 5 mm ATP, 5 mm MgCl2 in wash buffer. The Hsp70 ATPase domain was expressed in Escherichia coli and purified on ATP-agarose as previously described for Hsp70 (14Guerriero V. Raynes D.A. Gutierrez J.A. J. Cell. Physiol. 1989; 140: 471-477Crossref PubMed Scopus (29) Google Scholar).Limited Proteolysis—Assays were performed at room temperature with selected enzymes at a ratio of 1:250 (protease:protein) for a single protein and 1:125 when two proteins were combined so that the molar concentration of the protease remained relatively constant. The reaction mixtures were stopped with a protease inhibitor, incubated briefly on ice, and either snap frozen in liquid nitrogen for mass spectrometric analysis or prepared for other procedures by adding SDS sample buffer and heating to 95 °C for 5 min. Preincubation conditions were 15 min at 37 °C followed by 10 min at room temperature. All procedures used gave the same results as monitored by Coomassie Blue-stained gels. There were differences in the procedures for the data presented. For Edmund degradation, 12 μm HspBP1 was used in buffer A (0.5 m NaCl, 5 mm imidazole, and 20 mm Tris-HCl, pH 8.0) with CaCl2 (0.1 or 0.01 m) added to stabilize the proteases. The protease inhibitor was an agarose-immobilized trypsin inhibitor that was pelleted and removed before the sample was heated. For other analyses, proteins were used at a concentration of 24 μm, the reactions were carried out in buffer B (10 mm Tris-HCl, pH 7.5, 10 mm NaCl, and 5 mm dithiothreitol), and the protease inhibitor phenylmethylsulfonyl fluoride (1.1 mm) was used.Protein Identification and Analysis—Western blot analysis was performed as previously described (7Raynes D.A. Guerriero V. J. Biol. Chem. 1998; 273: 32883-32888Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Primary antibodies used were anti-HspBP1 (BD PharMingen), anti-Hsp70/Hsc70 SPA812 (StressGen, Victoria, British Columbia, Canada) for detection of the Hsp70ATPase domain, anti-Hsp70/Hsc70 SPA 820 for detection of full-length Hsp70 and anti-His6 antibody (BD Biosciences Clontech, Palo Alto, CA).Samples for Edman degradation were run on a 12.5% SDS-PAGE gel, transferred to Immobilon-P polyvinylidene difluoride membrane (Millipore, Billerica, MA), and stained with Coomassie Brilliant Blue R to visualize the protein bands. The protein bands were excised and submitted for analysis to the Laboratory for Protein Sequencing and Analysis at the University of Arizona Department of Chemistry. Samples were submitted to five cycles of degradation using an ABI 477A system.Mass spectrometric analysis was performed by the Mass Spectrometry Facility at the University of Arizona, Department of Chemistry. Masses were initially acquired using a Bruker Reflex III MALDI/TOF. 1The abbreviations used are: MALDI/TOF, matrix-assisted laser desorption/time-of-flight; ESI-LC/MS, electrospray ionization-liquid chromatography/mass spectrometry. For subsequent analyses, ESI-LC/MS using a Finnigan LCQ (Thermo Finnigan, San Jose, CA) was employed. This system used a C18 column with a gradient going from 95% buffer C (2% acetonitrile, 0.1% trifluoroacetic acid, 98% H2O) and 5% buffer D (90% acetonitrile, 0.1% trifluoroacetic acid, 10% H2O) to 5% buffer C and 95% buffer D over 30 min. The software used to analyze the observed masses was Xcaliber (Thermo Finnigan). To assign the observed masses to specific proteolytic fragments, the software program MS-Digest 2P. R. Baker and K. R. Clauser, prospector.ucsf.edu. was used. All protein assignments were within the observed tolerances of the instrumentation used.Circular Dichroism—Circular dichroism spectra of His6-tagged recombinant HspBP1 and recombinant M84–359 were acquired using an AVIV 60DS (V4.1t) spectropolarimeter at 15 °C. The proteins were first exposed to 5 mm dithiothreitol in buffer A for 24 h and then dialyzed into 5 mm phosphate buffer, pH 7.5, over 48 h in the cold. The samples were then diluted to ∼10 μm. After analysis the concentration of protein in the samples was determined more accurately using the absorbance at 280 and the molar extinction coefficients. The final concentrations were 13.7 μm for HspBP1 and 12.6 μm for M84–359. Spectra were acquired using 0.5-nm steps from 200 to 260 nm and a 1-mm path length. Each data set represents three repetitions. A separate spectrum was generated for the buffer alone, and this was subtracted from the protein spectra. The α-helical content was predicted using the mean residual ellipticity ([θ] in degrees cm2 dmol—1residue—1) at 222 nm according to the following standard equation (15Chen Y.H. Yang J.T. Biochem. Biophys. Res. Commun. 1971; 44: 1285-1291Crossref PubMed Scopus (326) Google Scholar): Fraction of α-helical content = ([θ]222 + 2340)/30,000. The fraction was then multiplied by 100 and expressed as percent α-helical content.Renaturation of Luciferase—Assays to measure renaturation of luciferase in rabbit reticulocyte lysate were done as previously described (7Raynes D.A. Guerriero V. J. Biol. Chem. 1998; 273: 32883-32888Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The denatured luciferase was added to reticulocyte lysate in the presence or absence of increasing amounts of full-length HspBP1 or truncation mutant. At 90 min three aliquots of each sample were removed and assayed for luciferase activity. Activities were compared with luciferase renatured in the absence of added protein. All mutants were tested at least twice.Hsp70 Binding to HspBP1 and Truncation Mutants—1 nmol of truncated or full-length HspBP1 was added to 20 μl of a 50% slurry of TALON resin and buffer A. After a 15-min incubation on ice the resin was pelleted and the supernatant removed. To the resulting resin pellet, 50 μl of reticulocyte lysate containing an ATP regenerating system (16Schumaker R.J. Hurst R. Sullivan W.P. McMahon N.J. Toft D.O. Matts R.L. J. Biol. Chem. 1994; 269: 9493-9499Abstract Full Text PDF PubMed Google Scholar) was added. After a 90-min incubation on ice with frequent gentle mixing, the resin pellet was quickly washed four times with ice-cold buffer C (10 mm Tris-HCl, pH 7.9, 1 mm MgCl2, 50 mm KCl, 7.5 mm imidazole, and 0.2% Tween). After the last wash the pellet was taken to dryness, and SDS sample buffer was added and heated to 95 °C for 5 min. The samples were then Western blotted as described above.ATPase Binding to HspBP1 and Truncation Mutants—Incubation of test proteins with the TALON resin, incubation conditions, and washing steps are all as described above for binding of full-length Hsp70. 35S-labeled ATPase domain was prepared using the TNT T7 quick-coupled transcription/translation system (Promega, Madison, WI) and [35S]methionine (Amersham Biosciences). The resulting 35S-labeled ATPase domain was diluted (1:2.67) with reticulocyte lysate containing an ATP regenerating system. 50 μl of this mixture was incubated with the TALON resin and test protein. The results were monitored using autoradiography.RESULTSThe Genomic Sequence of HspBP1 Predicts Seven Protein Domains—As a starting point for our studies, we first constructed a model of HspBP1 using the exon boundaries as deduced from the genomic sequence to divide the protein sequence into domains. This type of model of HspBP1 (Fig. 1A, model a) was constructed from information on the human genomic sequence (National Center for Biotechnology, www.ncbi.nlm.nih.gov), revealing that the HspBP1 protein is made from seven exons.HspBP1 Has One Structured Domain as Defined by Limited Proteolysis—To experimentally define the structural domains of HspBP1 and test the model that arose from the genomic sequence, we used limited proteolysis to probe HspBP1 higher order structure. Recombinant HspBP1 was subjected to proteolysis under mild conditions with three different proteases, trypsin, chymotrypsin, and proteinase K. All three proteases produced essentially the same results, so only the results for one, chymotrypsin, are shown (Fig. 1B). The proteases rapidly (∼5 min) cleaved HspBP1 to a stable fragment that has an apparent loss of 10 kDa from the parent compound. This protected fragment was stable for up to an hour in the presence of chymotrypsin and trypsin and for 30 min when proteinase K was used. Further degradation produced no discernable fragments. Western blot analysis with an antibody recognizing the His6 tag on the amino terminus showed that the amino terminus was not preserved in the stable fragment (data not shown). Western blot analysis with the HspBP1 antibody, a monoclonal raised against the most carboxyl third of HspBP1, indicated that this epitope was preserved in the stable fragment (data not shown).To identify the stable proteolytic fragment we employed Edman degradation and LC/MS to analyze samples of HspBP1 digested for 60 min with chymotrypsin. These analyses indicate that the stable proteolytic fragment encompasses amino acids 84–359. Five rounds of Edman degradation on the digested fragment produced the amino acid sequence RGQRE, which corresponds to amino acids 84–88 of the HspBP1 sequence. This is at a predicted chymotrypsin cleavage site. ESI-LC/MS of this same time point predicts a mass of 30,918 kDa, which corresponds to a fragment of amino acids 84–359 (predicted mass of 30,925 kDa). The spectrum for the proteolytic fragment is shown in Fig. 4B. Furthermore, a recombinant truncated version of HspBP1, M84–359, that had only the stable structured domain with an amino His6 tag, was expressed, purified, and subjected to proteolysis by chymotrypsin. After 60 min of exposure of M84–359 to chymotrypsin, the relative mobility was only slightly altered (Fig. 5C), possibly from removal of the His6 tag, further confirming that this part of HspBP1 is impervious to proteolysis.Fig. 4Representative MALDI/TOF and ESI-LC/MS spectra from limited proteolysis reactions.A, the mid-mass portion of the MALDI/TOF spectra of the Hsp70 ATPase domain preincubated with HspBP1 and then proteolyzed with chymotrypsin for 60 min. The full spectra and the assignment of masses to specific proteolytic fragments are given in Table I. B, the mass ion train for the major proteolytic fragment after 60 min of digestion of HspBP1 with chymotrypsin. The assigned positive charges are listed above the ions. The inset is a deconvolution of the data to give a major peak at 30,918 daltons, which corresponds to amino acids 84–359 of HspBP1. R.A., relative abundance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Limited proteolysis of HspBP1 domain II and the Hsp70 ATPase domain.A, Coomassie Blue-stained gel of the time course of limited proteolysis of M84–359 and the Hsp70 ATPase domain. The a indicates the full-length ATPase domain, b indicates the region where full-length M84–359 and new bands from the Hsp70 ATPase appear, and c indicates a new band only visible when the two proteins are preincubated together before proteolysis. B, Western blot of the samples in panel A with a polyclonal antibody recognizing the ATPase domain. Because it contains multiple epitopes, the band intensity does not indicate the protein amount. C, Western blot of the samples in panel A with an antibody to HspBP1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)From these analyses a new domain model of HspBP1 was constructed with two domains (Fig. 1A, model b). In this model the proteolytically stable structural domain is domain II, and the portion of HspBP1 removed by proteolysis is domain I. The region encoded by domain I in model b is similar to domain I in model a. From this point on, domain I will refer to amino acids 1–83, and domain II will refer to amino acids 84–359 as shown in model b.The Structural Conformation of HspBP1 Is Predicted to be 35% Helical—Structural prediction programs suggest that HspBP1 has large helical regions. To test these predictions we have used circular dichroism to analyze the helical content of HspBP1 and the recombinant version of the stable proteolytic fragment, M84–359 (Fig. 2). Analysis of these data resulted in a predicted conformation (including amino-terminal His6 tags) for HspBP1 that is 35% helical and for the stable proteolytic fragment M84–359 that is 43% helical.Fig. 2Circular dichroism of full-length HspBP1 and domain II. Mean residual ellipticity [θ] in degrees cm2 dmol—1 residue—1, for His6-tagged HspBP1 and M84–359 (which encompasses domain II). □, HspBP1; ▪, M84–359.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Association with HspBP1 Renders the Hsp70 ATPase Domain Susceptible to Limited Proteolysis—Limited proteolysis of HspBP1 and the Hsp70 ATPase domain was performed to assess the interaction of the two proteins. As previously reported, the Hsp70 ATPase domain alone was not digested by a 60-min treatment with chymotrypsin (Ref. 17Chappell T.G. Konfort B.B. Schmid S.L. Braell W.A. Rothman J.E. J. Biol. Chem. 1987; 262: 746-751Abstract Full Text PDF PubMed Google Scholar and data not shown). Preincubation of the Hsp70 ATPase domain with HspBP1, on the other hand, renders it susceptible to proteolysis (Fig. 3, A and B). It also appears to slow down the proteolysis of HspBP1 somewhat as shown in Fig. 3C but does not change the proteolytic sites. Preincubation on ice or at room temperature, as well as the addition of 3 mm MgCl2,50mm KCl, or 0.5 mm ATP (either to the ATPase domain prior to HspBP1 addition or at the time of addition) did not change the outcome of proteolysis as assessed by Coomassie Blue-stained gels of samples after 60 min of digestion (data not shown). The identity of the protein fragments was determined by mass spectrometry of limited proteolysis reactions after 5 and 60 min of proteolysis. Samples were analyzed using MALDI/TOF and ESI-LC/MS. Representative spectra are shown in Fig. 4. Fig. 4A shows the mid-mass range of a MALDI/TOF spectrum of a mixture of HspBP1 and the Hsp70 ATPase domain after 60 min of proteolysis. The full data from this analysis are given in Table I.Fig. 3Limited proteolysis of HspBP1 and the Hsp70 ATPase domain.A, a Coomassie Blue-stained gel of the time course of limited proteolysis of HspBP1 and the Hsp70 ATPase domain. The bracketed region indicated by the a shows where full-length HspBP1 and the ATPase domain run in the gel. The bracketed region next to the b indicates the region containing both domain II after digestion of HspBP1 under similar conditions and new bands from the Hsp70 ATPase domain. c indicates a new band only visible when the two proteins are preincubated together before proteolysis. B, Western blot of the samples in panel A with a polyclonal antibody recognizing the ATPase domain. Because it contains multiple epitopes, the band intensity does not indicate the protein amount. C, Western blot of the samples in panel A with an antibody to HspBP1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IMS analysis of proteolytic fragments from chymotrypsin digestion of HspBP1 preincubated with the Hsp70 ATPase domainListed are the observed fragments from MALDI/TOF analysis of a sample after 60 min of digestion. In the first column the assignment of the fragment is given, with A indicating the Hsp70 ATPase domain, B indicating HspBP1, followed by the corresponding amino acid numbers. Mobs is the observed mass in kDa., z is the charge of the observed ion, and % error is the difference between Mobs and the actual mass expressed as a percent of the actual mass. The results of ESI-LC/MS analysis performed on the same sample are given to provide confirmation and higher accuracy determination of larger masses. The columns to the right indicate which other 60-min samples contained the same proteolytic fragment. A is the HSP70 ATPase domain alone; B is HspBP1 alone, and C is M84-359 alone. AC is the Hsp70 ATPase domain preincubated with M84-359.AssignmentMALDI/TOFESI-LC/MSOther samples containing fragmentMobsz%ErrorMobs%ErrorABCACA 108-1838234108236-0.02xA 93-18399051-0.06A 79-183115341-0.0211531-0.04A 74-183121331-0.0412134-0.03A 69-183127181-0.0312716-0.05xA 184-371211471-0.0121144-0.02xA 184-371211402-0.04B 84-359308791-0.1330918-0.02xxxB 74-359317351-0.08A 69-371337451-0.37B 14-359377871-0.3437906-0.03A 2-382417701-0.2741885-0.01xx Open table in a new tab As shown in Table I, the areas of the ATPase domain that were susceptible to proteolysis after interacting with HspBP1 were determined to be concentrated mainly in subdomain I but also include the carboxyl terminus. The protected fragment left after proteolysis for 60 min (indicated by c in Fig. 3, A and B) was identified as amino acids 184–371 of the ATPase domain. This protected fragment encompasses the carboxyl terminal half of the ATPase domain with the exception of the last 11 amino acids. These results indicate that the association of the Hsp70 ATPase domain with HspBP1 dramatically changes the conformation of the ATPase domain.Domain II Alone Is Capable of Rendering the Hsp70 ATPase Domain Susceptible to Limited Proteolysis—Limited proteolysis experiments were performed after preincubating M84–359 with the Hsp70 ATPase domain. The results, shown in Fig. 5, are similar to the results when full-length HspBP1 was employed (note the appearance of bands indicated by b and c in Figs. 3B and 5B). ESI-LC/MS of a 60-min sample produced a similar pattern of Hsp70 ATPase fragments, as shown in the last column of Table I. These results indicate that domain II is sufficient to alter the conformation of the Hsp70 ATPase domain.Neither Domain II nor Other HspBP1 Mutants Are Capable of Fully Recapitulating the Inhibition of Luciferase Refolding of HspBP1—Next we sought to evaluate whether domain II or other HspBP1 truncation mutants were sufficient for other HspBP1 activities. The initial characterization of HspBP1 showed that it was a potent inhibitor of luciferase renaturation in reticulocyte lysate (7Raynes D.A. Guerriero V. J. Biol. Chem. 1998; 273: 32883-32888Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). To determine which part of HspBP1 was necessary for this function, we tested the ability of five mutants to inhibit luciferase renaturation in reticulocyte lysate. For these experiments cDNAs for truncation mutants of HspBP1 were constructed, expressed, and purified. One of these mutants, M1–138. was truncated at the carboxyl terminus of HspBP1 at amino acid 138. This mutant fully encompasses domain I (amino acids 1–83). The other four mutants are truncated from the amino terminus and are as follows: M51–359 starting at amino acid 51; M70–359 starting at amino acid 70, which begins with the first amino acid encoded by exon 1; the aforementioned M84–359, which is domain II only; and M301–359, which starts at amino acid 301. The results of these assays are shown in Fig. 6. Although the amino truncation mutants M51–359, M70–359, and M84–359 demonstrated the ability to inhibit luciferase renaturation, none of the mutants was as potent as HspBP1. One explanati
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