Multiple Domains of the Co-chaperone Hop Are Important for Hsp70 Binding
2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês
10.1074/jbc.m314130200
ISSN1083-351X
AutoresPatricia E. Carrigan, Gregory M. Nelson, Patricia J. Roberts, Jha'Nae Stoffer, Daniel L. Riggs, David F. Smith,
Tópico(s)Insect and Pesticide Research
ResumoThe Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction. The Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction. Native interactions of the major molecular chaperones Hsp70 and Hsp90 with known client proteins typically involve several attendant co-chaperone proteins. Among the activities attributed to partner co-chaperones are regulation of chaperone ATPase activity or nucleotide exchange (1Prodromou C. Pearl L.H. Curr. Cancer Drug Targets. 2003; 3: 301-323Crossref PubMed Scopus (232) Google Scholar, 2Mayer M.P. Brehmer D. Gassler C.S. Bukau B. Adv. Protein Chem. 2001; 59: 1-44Crossref PubMed Scopus (134) Google Scholar), recruitment of additional chaperones and co-chaperones (3Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), targeting of the client complex for degradation (4Hohfeld J. Cyr D.M. Patterson C. EMBO Rep. 2001; 2: 885-890Crossref PubMed Scopus (286) Google Scholar), or subcellular localization (5Pratt W.B. Silverstein A.M. Galigniana M.D. Cell Signal. 1999; 11: 839-851Crossref PubMed Scopus (149) Google Scholar). A commonly studied chaperone/client system is the assembly pathway for steroid receptor complexes in which multiple chaperones and co-chaperones participate in a multistep, dynamic assembly process (6Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1522) Google Scholar, 7Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1249) Google Scholar). The co-chaperone Hop, which can bind both Hsp70 and Hsp90, plays a key role in recruiting Hsp90 to preexisting receptor-Hsp70 complexes (3Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 8Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 9Kosano H. Stensgard B. Charlesworth M.C. McMahon N. Toft D. J. Biol. Chem. 1998; 273: 32973-32979Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), thus promoting assembly progression and ultimate functional maturation of the receptor.Similar to many Hsp70 and Hsp90 co-chaperones, tetratricopeptide repeat (TPR) 1The abbreviations used are: TPR, tetratricopeptide repeat; GST, glutathione S-transferase; RL, reticulocyte lysate; DOC, deoxycorticosterone; PR, progesterone receptor; GR, glucocorticoid receptor. 1The abbreviations used are: TPR, tetratricopeptide repeat; GST, glutathione S-transferase; RL, reticulocyte lysate; DOC, deoxycorticosterone; PR, progesterone receptor; GR, glucocorticoid receptor. domains of Hop mediate binding to Hsp (10Lassle M. Blatch G.L. Kundra V. Takatori T. Zetter B.R. J. Biol. Chem. 1997; 272: 1876-1884Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 11Chen S. Prapapanich V. Rimerman R.A. Honore B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar). Whereas the Hsp-binding co-chaperones most often contain a single TPR domain, Hop contains three distinct TPR domains. The C-terminal region of cytoplasmic forms of Hsp70 and Hsp90, both of which terminate with the amino acid sequence EEVD, have been implicated as the binding sites for Hop and for some other TPR co-chaperones (12Carrello A. Ingley E. Minchin R.F. Tsai S. Ratajczak T. J. Biol. Chem. 1999; 274: 2682-2689Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Russell L.C. Whitt S.R. Chen M.S. Chinkers M. J. Biol. Chem. 1999; 274: 20060-20063Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar); however, some TPR co-chaperones clearly interact with other Hsp regions (15Hohfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 16Prapapanich V. Chen S. Toran E.J. Rimerman R.A. Smith D.F. Mol. Cell. Biol. 1996; 16: 6200-6207Crossref PubMed Scopus (59) Google Scholar). Since Hop contains multiple TPR domains, the opportunity exists for Hop to simultaneously bind the EEVD sites of both Hsp70 and Hsp90.In a major advance toward understanding how TPR domains interface with the Hsp EEVD region, co-crystal structures were obtained for either of two Hop TPR domains complexed with an EEVD-containing peptide (14Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar). One co-crystal contained the N-terminal TPR domain of Hop, TPR1, and the octapeptide GPTIEEVD that corresponds to the Hsp70 C terminus. The other co-crystal contained one of the central TPR domains, TPR2a, in complex with the pentapeptide MEEVD that corresponds to the C-terminal sequence of Hsp90. Similar to some other reported TPR domains, both of the Hop TPR domains consisted of an antiparallel α-helical stack that forms a large groove along one surface of the domain. The EEVD-containing peptides lodged within this groove but in distinct orientations that related to TPR side-chain differences within the groove and unique amino acids in either peptide. In both co-crystal structures, basic TPR side chains within the groove formed salt bridges with acidic peptide side chains, forming what was termed the carboxylate clamp. Consistent with the TPR2a co-crystal structure, point mutation of a conserved carboxylate clamp position in any of several Hsp90-binding TPR co-chaperones has been shown to disrupt Hsp90 binding (13Russell L.C. Whitt S.R. Chen M.S. Chinkers M. J. Biol. Chem. 1999; 274: 20060-20063Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar).The co-crystal results suggest a straightforward model for an Hsp70-Hop-Hsp90 complex in which the C terminus of Hsp70 binds TPR1 of Hop and the C terminus of Hsp90 binds TPR2a of Hop. However, experimental results suggest that additional Hop domains influence Hsp binding. For instance, mutations within the C-terminal DP-domain of Hop greatly inhibit Hsp70 binding (3Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 17Nelson G.M. Huffman H. Smith D.F. Cell Stress Chaperones. 2003; 8: 125-133Crossref PubMed Scopus (37) Google Scholar), and deletions within TPR2b, the third TPR domain of Hop, can reduce Hsp90 binding (18Chen S. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (166) Google Scholar). Furthermore, as shown below, truncation of the EEVD from Hsp70 had little effect on Hop binding. To help resolve relevant interactions between full-length proteins, we have taken advantage of crystallographic structures and recent insight from sequence comparisons between Hop regions to design novel Hop point mutants that were assessed for Hsp binding, support of steroid receptor function in vivo, and conformational changes.EXPERIMENTAL PROCEDUREScDNA Mutagenesis and Construction of Expression Plasmids—A series of Hsp70 C-terminal truncation mutants was generated by introducing stop codons into the rat Hsc70 cDNA. Mutant cDNAs were generated by site-directed mutagenesis (QuikChange™ kit, Stratagene, La Jolla, CA) using the in vitro expression plasmid rat Hsc70/pSPUTK as template. The following mutants were generated, named according to the final amino acid encoded: N642, N614, N608, N604, N595, N575, N534, and N425.Hop point mutations were generated in a similar manner using human Hop/pSPUTK as template. Mutations in the following domains were generated: TPR1, Y27A, K73E, and K73A; TPR2a, K301E, K301A, R305E, and R305A; TPR2b, K429E, K429A, R433E, and R433A; DP1, D140A and D149A; AP1, D140A/D149A. A mutant in the DP2 domain (AP2) was constructed previously (3Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Sequences for mutagenic primers are available on request. All mutant cDNA sequences were verified by automated sequencing.For bacterial expression of recombinant proteins, mutant and wild type cDNA were cloned into pET28 for expression of untagged proteins in bacteria, pET30 for expression of His-tagged proteins, and pGEX-5X-3 for expression of GST-tagged proteins. When mutagenesis or PCR products were employed for subcloning, all insert sequences were verified by automated sequencing.In Vitro Binding Assays—Radiolabeled Hsp70 or Hop forms were generated by in vitro expression (TNT Kit; Promega, Madison, WI) in the presence of [35S]methionine. A 5-μl aliquot of each synthesis reaction was separated by SDS-PAGE and visualized by autoradiography; the labeled products were quantitated by densitometry (Fluor-S Multi-Imager; Bio-Rad). Molar equivalents of each radiolabeled product were added to rabbit reticulocyte lysate (1:1 lysate, Green Hectares, Oregon, WI) for immunoprecipitation and receptor assembly trials. Mouse monoclonal antibodies specific for Hop (F5), Hsp70 (BB70), or Hsp90 (H90-10) were preadsorbed to protein G-Sepharose (1 μg of antibody/μl of packed resin). For each immunoprecipitation reaction, 100 μl of reticulocyte lysate (RL) supplemented with a radiolabeled product were added to a 10-μl immunoresin pellet. The mixtures were incubated for 30 min at 30 °C with brief vortexing every 5 min. Resin-bound complexes were washed four times in 1 ml of wash buffer (20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 0.5% (v/v) Tween 20), resuspended in 20 μl of 2× SDS-PAGE sample buffer, and separated by SDS-PAGE. Gels were Coomassie-stained to visualize total proteins and then dried and autoradiographed to visualize bound radiolabeled proteins.Comparisons of wild type and mutated Hop forms assembling with PR complexes were performed as described previously (3Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Briefly, recombinant chicken PR was immunopurified from Sf9 insect cell extracts using monoclonal antibody PR22 preadsorbed to protein A-Sepharose. Each assembly reaction contained 10 μl of PR resin (representing 1 μg of PR) and an equimolar amount of a radiolabeled Hop form in 200 μl of RL (supplemented with an ATP-regenerating system to promote assembly reactions and 20 μg/ml geldanamycin to favor recovery of intermediate PR complexes containing Hop). Samples were incubated 30 min at 30 °C with brief vortexing at 5-min intervals to resuspend resin. Resin-bound complexes were washed three times in 1 ml of wash buffer, and bound proteins were separated by SDS-PAGE. Gels were stained with Coomassie Blue followed by autoradiography of dried gels. Bands on x-ray film were quantitated by densitometry.Yeast Strains and Plasmids—All Saccharomyces cerevisiae strains were generated in the W303a background (MATa leu2-112 ura3-1 trp1-1 his3-11,15 ade2-1 can1-100 GAL SUC2). Plasmids were introduced into yeast using the lithium acetate/polyethylene gycol protocol. Two to four independent transformants of each construction were analyzed. Strains were propagated in selective media consisting of 0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, the appropriate synthetic complete supplement mixture (Q-biogene, Carlsbad, CA), and 1.6% (w/v) agar for plates.To generate the Sti1-minus yeast strain (sti1Δ0), the entire coding region of STI1 was replaced with the Schizosaccharomyces pombe HIS5 gene flanked by loxP sites; the His marker was subsequently removed by transformation with a plasmid encoding the Cre recombinase (19Gueldener U. Heinisch J. Koehler G.J. Voss D. Hegemann J.H. Nucleic Acids Res. 2002; 30: e23Crossref PubMed Scopus (745) Google Scholar). Gene deletion was confirmed by yeast colony PCR, and the absence of Sti1p was confirmed by Western blot analysis with the anti-Sti1 monoclonal antibody ST2 (provided by D. Toft).As described previously (20Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (273) Google Scholar), the glucocorticoid receptor (GR) expression strain contained a pG/N795 plasmid constitutively expressing rat GR and the GRE-lacZ reporter plasmid pUCΔSS-26X. Wild type or mutant Hop cDNA was introduced into a yeast expression vector by ligating a HindIII/EcoRV fragment from pSPUTK into the constitutive expression plasmid p425GPD, which contains a LEU2 nutritional marker, a 2μ origin of replication, and a constitutive GPD transcriptional promoter.Yeast Assays for Hormone-induced Reporter Gene Expression—Hormone induction assays were conducted as previously described (20Riggs D.L. Roberts P.J. Chirillo S.C. Cheung-Flynn J. Prapapanich V. Ratajczak T. Gaber R. Picard D. Smith D.F. EMBO J. 2003; 22: 1158-1167Crossref PubMed Scopus (273) Google Scholar). Briefly, yeast strains were grown in selective media at 25 °C to an A600 of 0.05–0.12 units. Growth was monitored by spectrophotometry for 30 min before hormone addition to ensure that the culture was in exponential phase. Deoxycorticosterone (DOC) was added to the culture at 50 nm final concentration. To assay for β-galactosidase activity, 100 μl of culture was withdrawn and immediately added to 100 μl of the Gal-Screen™ substrate (Tropix, Bedford, MA) in 96-well microtiter plates at room temperature. Samples were taken at 10-min intervals until 70–80 min after hormone addition. The plate was read in a luminometer 2 h after the last sample was collected.To determine the rate of reporter expression, β-galactosidase induction curves were first generated by plotting relative light units against the A600 of the culture sample. Regression analysis of the linear portion of each data set yielded a best fit line (typically R2 > 0.98) whose slope is the growth-normalized rate of β-galactosidase expression.Yeast Cell Extracts and Western Immunoblots—To prepare whole cell extracts, washed cell pellets were resuspended in cracking buffer (8Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholarm urea, 5% (w/v) SDS, 40 mm Tris-HCl, pH 7.5, 0.1 mm EDTA, 0.04% (w/v) bromphenol blue) at 4 ml/g of cells. Cells were homogenized with glass beads in a Mini Bead Beater (Biospec Products, Bartlesville, OK). Cell homogenates were centrifuged to remove insoluble material and heated at 95 °C for 5 min. Lysate aliquots (10–15 μl) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immunostained for Sti1p (mouse monoclonal IgG ST-2), Hop (mouse monoclonal IgG F5), or the yeast ribosomal protein L3 (mouse monoclonal IgG anti-L3).Recombinant Protein Expression and Purification—BL21 bacterial cells were transformed with each of the various expression plasmids for protein expression. Briefly, 1 liter of LB medium plus 100 μg/ml ampicillin was inoculated 1:100 with an overnight culture of transformed bacteria. Cells were grown at 37 °C until the A600 reached 0.5, at which time 100 mm isopropyl β-d-galactopyranoside was added to a final concentration of 0.4 mm. Cells were incubated for an additional 3 h at room temperature. Bacterial cell pellets were resuspended in 10 ml of buffer A (20 mm Tris, pH 7.5, 1 mm EDTA, and 5 mm dithiothreitol plus Complete Mini protease inhibitor mixture (Roche Applied Science)) and sonicated to generate cell extracts.His-tagged proteins were purified by metal affinity chromatography according to manufacturer's instructions (Qiaexpressionist Kit, Qiagen, Valencia, CA). Extracts were applied to Ni2+-nitrilotriacetic acid affinity resin and incubated for 1 h at 4 °C. Resins were washed three times in 1 ml wash buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, and 20 mm imidazole). Protein was eluted at 250 mm imidazole.GST-tagged bacterial extracts were rocked batch-wise with glutathione-Sepharose 4B resin (Amersham Biosciences) for 1 h at 4 °C. Proteins were eluted from the glutathione resin with 50 mm Tris-HCl containing 10 mm reduced glutathione.Untagged Hop forms were purified by three-step chromatography (Akta FPLC, Amersham Biosciences). First, bacterial extracts containing untagged proteins were loaded on a HiPrep16/10 Heparin-Sepharose column equilibrated in buffer A, and proteins were eluted with a salt gradient of 0–0.5 m KCl in buffer A. Peak fractions containing Hop were identified by gel electrophoresis and pooled. For the next purification step, samples were loaded on a Resource Q column and eluted with 0–500 mm KCl. Peak fractions were again pooled and then applied to a 16/60 Superdex 200 column equilibrated with 20 mm Tris, pH 7.5, 250 mm KCl, and 5 mm MgCl2. Final peak fractions for Hop were pooled and concentrated using Amicon Centricon-10 filters (Millipore Corp., Billerica, MA). The purity of all Hop forms was judged to be greater than 95%.GST-Hop Pull-down Assays—Previously purified and quantitated GST-tagged Hop forms were incubated batch-wise with glutathione-Sepharose 4B (5 μg of protein plus 20 μl of bed volume resin) for 30 min at room temperature on a rocking platform and washed three times in 1 ml of incubation buffer (20 mm Tris, pH 7.5, 100 mm KCl, 5 mm MgCl2, and 2 mm dithiothreitol). Resin pellets were resuspended in 150 μl of incubation buffer plus 10 μg of purified Hsp70 for 30 min at 30 °C. Resin was subsequently washed three times in 1 ml of ice-cold incubation buffer containing 0.01% Nonidet P-40. Bound proteins were eluted with SDS sample buffer, separated on gels, and visualized by Coomassie Blue staining.Protease Digestion of Recombinant Hop Forms—Purified His-tagged Hop forms were subjected to partial proteolytic analysis (21Konigsberg W.H. Methods Enzymol. 1995; 262: 331-346Crossref PubMed Scopus (14) Google Scholar) using chymotrypsin (Type VIII; Sigma) or subtilisin Carlsberg (Type VIII; Sigma). Digestion mixtures containing 30 μg of Hop and 300 ng of protease in 50 mm Tris-HCl, pH 8.0 (60-μl total volume) were incubated at 30 °C over a 60-min time course with the removal of 10-μl aliquots at 1, 5, 15, and 60 min. Proteolysis was quenched by the addition of 10 mm phenylmethylsulfonyl fluoride and/or by placing the sample in an acetone/dry ice bath. Protein fragments were separated on precast 4–20% gradient gel (Bio-Rad) and Coomassie-stained to visualize protease digestion patterns. Bands were quantitated by densitometry.RESULTSKnown structural/functional domains of Hsp90, Hsp70, and Hop are illustrated in Fig. 1A. Hsp90 contains an N-terminal ATPase domain (22Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar), a large middle domain (23Meyer P. Prodromou C. Hu B. Vaughan C. Roe S.M. Panaretou B. Piper P.W. Pearl L.H. Mol. Cell. 2003; 11: 647-658Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar), and a C-terminal region of unknown conformation that contains a strong dimerization site. There is evidence for peptide binding by both the ATPase and middle domains of Hsp90 (24Scheibel T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (230) Google Scholar). Hsp70 contains an N-terminal ATPase domain (25Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar) unrelated to the corresponding Hsp90 domain, a central peptide binding domain (26Morshauser R.C. Hu W. Wang H. Pang Y. Flynn G.C. Zuiderweg E.R. J. Mol. Biol. 1999; 289: 1387-1403Crossref PubMed Scopus (134) Google Scholar), and a C-terminal 10-kDa domain (27Chou C.C. Forouhar F. Yeh Y.H. Shr H.L. Wang C. Hsiao C.D. J. Biol. Chem. 2003; 278: 30311-30316Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Hop contains three TPR domains plus a C-terminal DP domain and a putative central DP domain (further described below). As indicated, TPR1 is thought to bind the C-terminal PTIEEVD sequence of Hsp70, whereas Hop TPR2a is thought to bind the C-terminal MEEVD sequence of Hsp90 (14Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar).In Fig. 1B, the TPR2a/MEEVD co-crystal structure is depicted in two alternate views. The TPR motifs in antiparallel α-helices form the peptide binding groove. Positively charged side chains of Arg305 and Lys301 that form the TPR2a carboxylate clamp are highlighted, as are the negatively charged side chains of the MEEVD peptide. The TPR1/GPTIEEVD co-crystal shows a similar overall structure, although the carboxylate clamp residues and peptide side chains are oriented in a unique manner.Hsp70 C-terminal Sequences Necessary for Hop Binding—To test directly which C-terminal sequences of Hsp70 are required for Hop binding, a series of C-terminal truncation mutants was prepared (Fig. 2A), and each mutant was compared with full-length Hsp70 in a Hop co-immunoprecipitation assay (Fig. 2B). The smallest truncation (N642) lacks the terminal EEVD site, the next truncation (N614) lacks sequences through the GGMP-repeat region (dashed underlined region), and additional mutants progressively remove upstream highly conserved sequence patches (solid underlined regions). Equivalent amounts of radio-labeled Hsp70 forms were added to rabbit reticulocyte lysate, and the Hsp90-Hop-Hsp70 complex was immunoprecipitated using an anti-Hop antibody. Proteins were separated by SDS gel electrophoresis and visualized by Coomassie staining (Fig. 2B, top panel) or autoradiography (middle panel). The amounts of radio-labeled Hsp70 added to each mixture are shown in an additional autoradiograph (bottom panel).Fig. 2Interaction of Hsp70 C-terminal truncation mutants with Hop.A, shown is the amino acid sequence for the C-terminal region (positions 587–646) of rat Hsc70 (PIR accession number P08109). The highlighted sequences are regions of highest conservation among Hsp70 forms (solid underline), the GGMP repeat motif that is found in some Hsp70 family members (dashed underline), and the octapeptide (boldface letters) used for co-crystallization with Hop TPR1. Stop codons were introduced in this region to generate a series of truncation mutants, as indicated. B, wild-type (WT) and mutant cDNA were used to generate radiolabeled protein products. Molar equivalents of each Hsp70 form (Input) were added to normal rabbit reticulocyte lysate, from which Hop complexes were immunoprecipitated. Immune complexes were separated by SDS gel electrophoresis and visualized by Coomassie staining for total proteins (top panel) or by autoradiography to detect bound radiolabeled Hsp70 forms (middle panel). Stained bands, which represent rabbit Hsp90 and Hsp70 that co-precipitate with Hop and the anti-Hop heavy chain (HC), are indicated on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Contrary to expectations, truncation of the C-terminal EEVD (N642) or more extensive truncations (N614 and N608) had little effect on recovery of Hsp70 in Hop complexes. Only when the final 40–50 amino acids were truncated (N604 and N595) was there a major loss of Hop binding. An additional truncation mutant that terminates at position 575 also failed to bind Hsp70 (not shown), thus minimizing the likelihood that N604 and N595 artifactually block Hop access to upstream binding sites. On the other hand, in probing the region from 590 to 606 that contains sequences necessary for Hop binding, several single and double point mutations were generated, but none of these inhibited Hop binding (not shown). Point mutant results argue against the presence of a discrete Hop binding site in this region. Although this limited mutational analysis does not identify sequences in the C-terminal region of Hsp70 that are sufficient for Hop binding, we can conclude that the EEVD and adjacent GGMP repeat region are not required for Hop binding in our assay.Mutagenic Analysis of Hop Domains—To further explore Hop/Hsp70 interactions, we generated domain-specific point mutants of Hop to verify that TPR1 is required and to test whether other Hop domains participate in Hsp70 binding. Careful sequence analysis of Hop revealed a previously overlooked symmetry between the N-terminal and C-terminal regions of Hop. Fig. 3 shows an alignment of TPR1 plus downstream sequences compared with TPR2b and downstream sequences. We have previously shown that sequences downstream from TPR2b contain a DP-repeat motif (underlined sites) that contributes to an independent structural domain (17Nelson G.M. Huffman H. Smith D.F. Cell Stress Chaperones. 2003; 8: 125-133Crossref PubMed Scopus (37) Google Scholar). Truncation or point mutations within the so-called DP domain lessens binding of Hop to Hsp70 without affecting Hop binding to Hsp90. As the alignment in Fig. 3 reveals, there is similarity between the C-terminal DP domain and sequences correspondingly positioned downstream from TPR1. We refer to this recently recognized N-terminal DP region as DP1 and the original DP domain as DP2.Fig. 3Sequence similarity between the N-terminal and C-terminal regions of Hop. In the alignment of amino acids 6–187 with 362–539 of human Hop (PIR accession number A38093), regions of similarity are observed between TPR1 and TPR2b (shaded box); the carboxylate clamp positions targeted for mutagenesis are indicated (bold-face type and underlined). The latter portion of each sequence contains a DP repeat motif (open box); the various DP elements are indicated (boldface type), as are residues targeted for mutagenesis (underlined). Alignments were generated using the Gap component of SeqWeb (version 2.1; Accelrys).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Point mutants were generated for each of the three TPR domains as well as the two DP regions. Within the TPR domains, point mutations targeted basic residues in the carboxylate clamp. For TPR2b, whose structure has not been solved, basic amino acids corresponding to the carboxylate clamp positions in TPR1 were mutated (double underlines in Fig. 3). In other Hsp90-binding TPR proteins, point mutations of basic amino acids that form the carboxylate clamp have been shown to efficiently disrupt Hsp90 binding (13Russell L.C. Whitt S.R. Chen M.S. Chinkers M. J. Biol. Chem. 1999; 274: 20060-20063Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 28Van Der Spuy J. Kana B.D. Dirr H.W. Blatch G.L. Biochem. J. 2000; 345: 645-651Crossref PubMed Scopus (38) Google Scholar, 29Ward B.K. Allan R.K. Mo
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