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• W2077307831 abstract "To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5′-(β,γ-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization. To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5′-(β,γ-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization. heat shock protein adenosine 5′-(β,γ-imino)triphosphate high performance liquid chromatography polyacrylamide gel electrophoresis. Heat shock proteins (HSPs)1 are ubiquitous proteins found in all organisms and cell compartments. They are involved in cellular functions as varied as protein synthesis and proteolysis (1Craig E.A. Baxter B.K. Becker J. Halladay J. Ziegelhoffer T. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 31-52Google Scholar), stress tolerance (2Parsell D.A. Lindquist S. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 457-494Google Scholar), protein translocation into mitochondria (3Gambill B.D. Voos W. Kang P.J. Miao B. Langer T. Craig E.A. Pfanner N. J. Cell Biol. 1993; 123: 109-117Crossref PubMed Scopus (222) Google Scholar) or the endoplasmic reticulum (4Brodsky J.L. Schekman R. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 85-109Google Scholar), and folding and assembly of proteins (5Craig E.A. Science. 1993; 260: 1902-1903Crossref PubMed Scopus (162) Google Scholar). Members of the HSP70 family are molecular chaperones that hold in common the ability to discriminate between unfolded polypeptide chains and native proteins. They participate in protein assembly by preventing aggregation due to hydrophobic interactions (6Hartl F.-U Hlodan R. Langer T. Trends Biochem. Sci. 1994; 19: 20-25Abstract Full Text PDF PubMed Scopus (357) Google Scholar). HSP70 family members are highly conserved proteins, and share a structural organization in three domains: a 44-kDa N-terminal ATPase domain (7Flaherty K.M. DeLuca-Flaherty C. McKay D. Nature. 1990; 346: 623-628Crossref PubMed Scopus (830) Google Scholar, 8Flaherty K.M. McKay D.B. Kabsch W. Holmes K.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5045-5401Crossref Scopus (297) Google Scholar, 9Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (701) Google Scholar), an 18-kDa C-terminal peptide-binding domain (10Wang T.-F. Chang J. Wang C. J. Biol. Chem. 1993; 268: 26049-26051Abstract Full Text PDF PubMed Google Scholar, 11Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1060) Google Scholar), and a less conserved C-terminal tail whose function and complete three-dimensional structure are unknown. The ATPase activity of HSP70 proteins is enhanced upon binding to synthetic peptides (12Flynn G.C. Chappel T.G. Rothman J.E. Science. 1989; 245: 385-390Crossref PubMed Scopus (580) Google Scholar, 13Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar). The size and nature of peptidic substrates required to fully stimulate the ATPase activity of HSP70 proteins have been determined to be 7-residue-long peptides, rich in aliphatic amino acids (14Flynn G.C. Pohl J. Flocco M.T. Rothman J.E. Nature. 1991; 353: 726-730Crossref PubMed Scopus (635) Google Scholar, 15Gragerov A. Zeng L. Zhao X. Burkholder W. Gottesman M.E. J. Mol. Biol. 1994; 235: 848-854Crossref PubMed Scopus (212) Google Scholar, 16Jordan R. McMacken R. J. Biol. Chem. 1995; 270: 4563-4569Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Detailed enzymological studies on monomeric, recombinant bovine Hsc70 revealed that binding of peptides increases the rate of ADP and inorganic phosphate release leading to an increase in the rate of ATP hydrolysis (17Takeda S. McKay D.B. Biochemistry. 1996; 35: 4636-4644Crossref PubMed Scopus (75) Google Scholar). In the same study, Takeda and McKay observed that the Mg.ADP.Hsc70 form has higher affinity for peptide than the Mg.ATP.Hsc70 form, in agreement with studies on Escherichia coli DnaK (18Landry S.J. Jordan R. McMacken R. Gierasch L.M. Nature. 1992; 355: 455-457Crossref PubMed Scopus (257) Google Scholar, 19Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (423) Google Scholar) and bovine brain Hsc70 (20Greene L.E. Zinner R. Naficy S. Eisenberg E. J. Biol. Chem. 1995; 270: 2967-2973Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). It was recently demonstrated that ATP-bound HSP70 proteins have high on/off rates of binding/release of substrates, whereas ADP-bound chaperones exhibit higher affinity for peptidic substrates through slower off rates (reviewed in Ref. 21Johnson J.L. Craig E.A. Cell. 1997; 90: 201-204Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Many HSP70 proteins are also regulated by accessory proteins. A well studied system is the E. coliDnaK chaperone, which is regulated by the nucleotide exchange factor GrpE, which increases the rate of ADP/ATP exchange (22Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (690) Google Scholar, 23McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (350) Google Scholar) and by the highly conserved DnaJ, which increases the rate of γ-phosphate cleavage (16Jordan R. McMacken R. J. Biol. Chem. 1995; 270: 4563-4569Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 22Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (690) Google Scholar). In addition to the different conformations HSP70 proteins adopt in the presence of substrates, they also self-associate into multiple oligomeric species that interconvert with each other (24Schlossman D.M. Schmid S.L. Braell W.A. Rothman J.E. J. Cell Biol. 1984; 99: 723-733Crossref PubMed Scopus (288) Google Scholar, 25Schönfeld H.-J. Schmidt D. Schroder H. Bukau B. J. Biol. Chem. 1995; 270: 2183-2189Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 26Benaroudj N. Batelier G. Triniolles F. Ladjimi M.M. Biochemistry. 1995; 34: 15282-15290Crossref PubMed Scopus (51) Google Scholar). In the bacterial system, addition of the dimeric cofactor GrpE to heterogeneous multimeric DnaK results in a slow conversion of oligomers into monomers and stabilization of (GrpE)2·DnaK complexes (25Schönfeld H.-J. Schmidt D. Schroder H. Bukau B. J. Biol. Chem. 1995; 270: 2183-2189Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Mammalian Hsp70 oligomers of high molecular weights do not behave like the dimers and trimers in that they do not dissociate upon ATP addition and are less thermostable (27Palleros D.R. Welch W.J. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5719-5723Crossref PubMed Scopus (280) Google Scholar). As the expression of most HSP70 proteins is increased upon heat shock treatment, the effect of temperature on self-association of HSP70 proteins has also been investigated. It was observed that an increase in temperature in the 40–47 °C range leads to self-association/aggregation of Hsp73 and Hsc70 (27Palleros D.R. Welch W.J. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5719-5723Crossref PubMed Scopus (280) Google Scholar, 28Benaroudj N. Fang B. Triniolles F. Ghelis C. Ladjimi M.M. Eur. J. Biochem. 1994; 221: 121-128Crossref PubMed Scopus (20) Google Scholar). These data emphasize the complexity of the HSP70 cycle and how unfolded peptidic substrates, adenine nucleotides, accessory proteins and oligomerization affect the conformation of the proteins. The mechanisms by which self-association and depolymerization might affect the functions and activities of the molecular chaperones in normal and stress conditions are poorly understood. GRP78/BiP is one of the two members of the HSP70 family involved in polypeptide translocation across the endoplasmic reticulum membrane of eukaryotic cells (29Panzner S. Dreier L. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 30Schlenstedt G. Harris S. Risse B. Lill R. Silver P.A. J. Cell Biol. 1995; 129: 979-988Crossref PubMed Scopus (134) Google Scholar, 31Craven R.A. Egerton M. Stirling C.J. EMBO J. 1996; 15: 2640-2650Crossref PubMed Scopus (143) Google Scholar). GRP78/BiP plays a role in the folding and assembly of secreted or membrane proteins as well as in their transport (32Gething M.J. Blond-Elguindi S. Mori K. Sambrook J.F. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 111-135Google Scholar, 33Little E. Ramakrishnan M. Roy B. Gazit G. Lee A.S. Crit. Rev. Eukaryotic Gene Exp. 1994; 4: 1-18Crossref PubMed Scopus (357) Google Scholar, 34Beggah A. Mathews P. Beguin P. Geering K. J. Biol. Chem. 1996; 271: 20895-20902Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 35Hendershot L. Wei J. Gaut J. Melnisk J. Aviel S. Argon Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5269-5274Crossref PubMed Scopus (131) Google Scholar). GRP78/BiP also binds transiently to early folding intermediates and dissociates rapidly (36Melnick J. Dul J.L. Argon Y. Nature. 1994; 370: 373-375Crossref PubMed Scopus (368) Google Scholar). Like the other members of the HSP70 family, BiP is a peptide-stimulated ATPase (12Flynn G.C. Chappel T.G. Rothman J.E. Science. 1989; 245: 385-390Crossref PubMed Scopus (580) Google Scholar, 13Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar). The spectrum of peptidic substrates recognized by BiP (37Blond-Elguindi S. Cwirla S.E. Dower W.J. Lipshutz R.J. Sprang S.R. Sambrook J.F. Gething M.J. Cell. 1993; 75: 717-728Abstract Full Text PDF PubMed Scopus (567) Google Scholar), although broad enough to allow binding to all nascent chains translocated across the endoplasmic reticulum membrane, is also highly specific; BiP does not bind to fully folded and assembled proteins, except to co-chaperones like the DnaJ yeast homologues Scj1 and Sec63 (30Schlenstedt G. Harris S. Risse B. Lill R. Silver P.A. J. Cell Biol. 1995; 129: 979-988Crossref PubMed Scopus (134) Google Scholar, 38Brodsky J.L. Schekman R. J. Cell Biol. 1993; 123: 1355-1363Crossref PubMed Scopus (227) Google Scholar). BiP has a specificity distinct from that of other HSP70 proteins (39Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1994; 269: 30470-30478Abstract Full Text PDF PubMed Google Scholar) and is not functionally interchangeable with other HSP70 proteins (40Wiech H. Buchner J. Zimmerman M. Zimmerman R. Jakob U. J. Biol. Chem. 1993; 268: 7414-7421Abstract Full Text PDF PubMed Google Scholar, 41Brodsky J.L. Hamamoto S. Feldheim D. Schekman R. J. Cell Biol. 1993; 120: 95-102Crossref PubMed Scopus (130) Google Scholar). BiP self-associates into dimers, trimers, and higher molecular weight oligomers (13Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar, 42Wei J. Hendershot L.M. J. Biol. Chem. 1995; 270: 26670-26676Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) that dissociate upon addition of millimolar concentrations of ATP (43Carlino A. Toledo H. Skaleris D. DeLisio R. Weissback H. Brot N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2081-2085Crossref PubMed Scopus (67) Google Scholar) or 0.1–0.2 mm specific peptides used as model for unfolded substrates (13Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar). Here we describe a system that enables us to investigate the role of each domain in the self-association equilibrium of recombinant murine BiP. By using an engineered protein that contains an enterokinase site at the junction between the N-terminal ATPase domain and the C-terminal peptide binding domain (FLAG-BiP.ent), we show that BiP is a flexible and malleable molecule that interchanges between different oligomeric conformations and degrees of polymerization in a slow, concentration- and temperature-dependent, process. Binding of ATP, or of a synthetic peptide, induces depolymerization of BiP oligomers into monomeric species at different rates. Upon enterokinase cleavage of monomeric FLAG-BiP.ent, the two domains dissociate and the C-terminal domain self-associates into multiple oligomeric species. When enterokinase cleavage is performed in the presence of a synthetic peptide, the C-terminal domain can be isolated as a mostly monomeric fragment. Addition of ATP during enterokinase treatment does not prevent self-association of the C-terminal fragment. Our data indicate that peptide-induced depolymerization of BiP is controlled by the peptide binding C-terminal domain, and that a signal is being transmitted to the ATPase domain upon BiP monomerization that results in an increase in the rate of ATP hydrolysis. This study emphasizes the role of interdomain interactions in the regulation of BiP activity and degree of oligomerization. The vector pSecB115, described previously (13Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar), was digested with SalI and religated with a double-stranded SalI linker encoding the FLAG epitope (DYKDDDDK), in frame with the N-terminal OmpT signal sequence that precedes the full-length murine GRP78/BiP gene in pSecB115. The two oligonucleotides used for the SalI linker were designed as follows: 5′-TCGACG GAC TAC AAG GAC GAC GAT GAC AAG-3′ and 5′-TCGA CTT GTC ATC GTC GTC CTT GTA GTC CG-3′ (the SalI cohesive ends are underlined). The resulting plasmid, pFLAG-BiP was then cut with BamHI, and the 2-kilobase pair fragment containing murine BiP/GRP78 cDNA (44Kozutsumi Y. Normington K. Press E. Slaughter C. Sambrook J. Gething M.-J. J. Cell Sci. Suppl. 1989; 11: 115-137Crossref PubMed Google Scholar) was subcloned in dephosphorylated double-stranded M13mp18 bacteriophage. Single-stranded DNA was purified, and loop-in mutagenesis was carried out with a non-coding mutagenic nucleotide, 5′-G TAC CAG ATC ACC TGT ATC CTT ATCGTCATC ATCACCAGAGAGGACACC-3′, which annealed with the 3′ end of the BiP ATPase domain, and the 5′ end of the C-terminal domain, and contains an internal extra sequence that encodes for an enterokinase site (underlined) to be inserted between the translated residues Asp-390 and Asp-392 in the murine GRP78/BiP sequence. Bacteriophage plaques were screened with the 5′-CCTTATCGTCATCATCACC-3′ probe, and the mutant BamHI cassette was subcloned in the pSecB115 vector to yield pFLAG-BiP.ent. The entire coding sequence was sequenced using Sequenase (U. S. Biochemical Corp.) with the following nucleotides as primers: pET12a (5′-GCC AGT CAC TAT GGC GTG-3′), P2 (5′-TCC AGG CCA TAT GCA ATA GC-3′), P3 (5′-GAA ATT TCT TCT GGG GC-3′), P4 (5′-CTT TTC TAC CTC ACG CCG-3′), P5 (CCT CAT CGG GGT TTA TG-3′), P6 (5′-GGA ATT CCA GTC AGA TC-3′), P7 (CCC AGC TTT TCT TTA TC-3′), and P8 (5′- GCT CTA GCA GAT CAG TG-3′). No additional mutations were identified. All experiments were carried out as described (45Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). BiP.ent cDNA was amplified from pFLAG-BiP.ent with the 5′ primer XhoI-5, 5′-CCGCTCGAG GAG GAG GAC AAG AAG GAG-3′, which introduces an XhoI site at the 5′ extremity of the BiP.ent sequence (underlined), and the reverse primer HindIII-3′, 5′-CCC AAGCTT TAA TGC AGT TTA TC-3′, which anneals at the 3′ end of the DNA, after the elements of transcription termination of the pET12a vector at the level of a uniqueHindIII site (underlined). The PCR product was cleaved withXhoI and HindIII and ligated into pET19b to yield pHis-BiP.ent. The same XhoI-5′ and HindIII-3′ primers were used to amplify the BiP sequence from the plasmid pSecBB115 (13Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar), and the XhoI-HindIII fragment was subcloned in pET19b to yield the plasmid pHis-BiP. The FLAG epitope allows purification on anti-FLAG M2 affinity gel (IBI, Inc.), thus minimizing the extent of contamination of BiP by bound adenine nucleotides. E. coliBL21(DE3) cells were transformed with pFLAG-BiP.ent. Cells were grown in M9 medium supplemented with 50 μg/ml carbenicillin. T7 RNA polymerase expression was induced at A 600 nm ∼ 0.8 by addition of 0.4 mm (final concentration) of isopropyl-1-thio-β-d-galactopyranoside. After a 1-h induction period, the cells were harvested by centrifugation for 10 min at 5,000 rpm at 4 °C, resuspended in Buffer A (20 mmTris-HCl, pH 8.0, 175 mm KCl, 5 mmMgCl2), and broken in a French pressure cell (10,000 p.s.i. at 4 °C). Total cell extracts were centrifuged at 4 °C for 45 min at 45,000 × g. The supernatant was loaded at a flow rate of 2 ml/min on a column packed with 23 ml of anion exchanger Source 15Q medium (Amersham Pharmacia Biotech), equilibrated in Buffer A, and connected to an FPLC system (Amersham Pharmacia Biotech). The elution was performed with a 175–325 mm KCl linear gradient. Fractions containing FLAG-BiP.ent (230–270 mmKCl) were pooled and loaded onto an M2 column equilibrated in Buffer B (50 mm Tris, pH 7.4, 150 mm KCl). After removal of the unbound proteins by extensive washes with Buffer B, FLAG-BiP.ent was eluted with 0.1 m glycine-HCl, pH 3.5, and immediately neutralized with 1 m Tris, pH 8.0. To remove bound nucleotides, 4 mm EDTA was added to the sample and proteins were precipitated by ammonium sulfate (75% of saturation) as described for Hsc70 (24Schlossman D.M. Schmid S.L. Braell W.A. Rothman J.E. J. Cell Biol. 1984; 99: 723-733Crossref PubMed Scopus (288) Google Scholar). When necessary, FLAG-BiP.ent was further purified on HR10/30 Superose 12 (Amersham Pharmacia Biotech). The sample was then dialyzed against Binding Buffer (20 mm Hepes, pH 7.0, 75 mm KCl, 5 mm MgCl2) or Buffer C (20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 2 mm CaCl2) for enterokinase cleavage. His-tagged proteins were expressed in BL21(DE3) cells (46Studier W.F. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar), and purified by affinity chromatography on His.Bind resin N.D. as described by the manufacturer (Novagen, Inc.). When needed, further purification was performed on Superose 12 HR 10/30 as indicated in the text. Bound nucleotides were removed as described (24Schlossman D.M. Schmid S.L. Braell W.A. Rothman J.E. J. Cell Biol. 1984; 99: 723-733Crossref PubMed Scopus (288) Google Scholar). All purified proteins were exhaustively dialyzed against Binding Buffer. Peptide pep2 (LSVKFLT) was identified as a BiP substrate by affinity panning of a library of Trp-less, Met-less peptides displayed on bacteriophages 2S. Y. Blond and M.-J. Gething, unpublished results. using procedures very similar to those previously described for the identification of a large series of other BiP-binding peptides (37Blond-Elguindi S. Cwirla S.E. Dower W.J. Lipshutz R.J. Sprang S.R. Sambrook J.F. Gething M.J. Cell. 1993; 75: 717-728Abstract Full Text PDF PubMed Scopus (567) Google Scholar). The synthesis of pep2 was performed on a 431 peptide synthesizer from Applied Biosystems Inc (University of Illinois at Chicago Resource Center). The coupling agents were 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate and 1-hydroxybenzotriazole (47Knorr R. Trzeciak A. Bannwarth W. Gillessen D. Tetrahed. Lett. 1989; 30: 1927-1930Crossref Scopus (932) Google Scholar). Pep2 was purified by high performance liquid chromatography (HPLC) on a semi-preparative C8 column and analyzed by amino acid analysis and mass spectrometry (Protein Research Laboratory, University of Illionois at Chicago). Concentrations of the peptide stock solutions were determined as follows: Pep2 was hydrolyzed with 6 n HCl and then derivatized with phenylisothiocyanate using the Millipore-Water Pico-Tag work station, and analyzed by HPLC using the ABI 130A HPLC separation system with an ABI C-18 Brownlee column (2.1 × 220 mm) at 37 °C. The amino acid standards used to calculate the peptide concentration were purchased from Pierce (catalog no. 27300). We engineered plasmid pFLAG-BiP.ent that allows BiP expression in E. coli and has the following features: an N-terminal OmpT signal sequence to target the protein into the periplasmic space (46Studier W.F. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar), followed by a FLAG sequence (DYKDDDDK), recognized by the M2 anti-FLAG monoclonal antibody (IBI), and which also specifies a recognition site for enterokinase (DDDDK), and finally the full-length murine BiP (44Kozutsumi Y. Normington K. Press E. Slaughter C. Sambrook J. Gething M.-J. J. Cell Sci. Suppl. 1989; 11: 115-137Crossref PubMed Google Scholar). A second enterokinase site was inserted by loop-in mutagenesis between the N-terminal ATPase domain and the C-terminal domain C30, at position D390, as described under “Materials and Methods.” FLAG-BiP.ent was expressed in BL21(DE3) cells upon isopropyl-1-thio-β-d-galactopyranoside induction, immobilized onto an anti-FLAG M2 affinity Gel (IBI), and eluted from the column through reversible denaturation in acidic buffer as described under “Materials and Methods.” FLAG-BiP.ent migrates on a denaturing polyacrylamide gel as an 80-kDa species, in agreement with its expected molecular weight. Circular dichroism spectroscopy analysis shows a significant amount of secondary structure: 35–40% α-helix, 40–45% β-sheet, 5% β-turn, and 8–12% random-coil as determined with SELCON software (48Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (946) Google Scholar). These results are similar to those reported for Hsc70 (49Sadis S. Raghavendra K. Hightower L.E. Biochemistry. 1990; 29: 8199-8206Crossref PubMed Scopus (34) Google Scholar, 50Park K. Flynn G. Rothman J.E. Fasman G. Protein Sci. 1993; 2: 325-330Crossref PubMed Scopus (25) Google Scholar). To address whether the N-terminal FLAG extension and the presence of an enterokinase site at the junction between the catalytic domain and the peptide binding domain would affect the catalytic properties of BiP, we engineered the plasmids pHis-BiP.ent and pHis-BiP and characterized the catalytic properties of purified recombinant His- BiP.ent and His-BiP proteins. Michaelis and catalytic constants were determined in steady state conditions for FLAG-BiP.ent, His-BiP.ent and His-BiP, in the absence and presence of a synthetic peptide (pep2: LSVKFLT), and found to be in the same range of values reported for unmodified murine BiP and other HSP70 proteins (Table I). Our data indicate that the addition of an N-terminal FLAG epitope or histidine tag, as well as the insertion of an enterokinase site at the junction between the two domains do not profoundly alter the overall secondary structure of BiP, nor its ability to bind ATP or peptides with reasonable affinities. However, the basal ATPase activity of both FLAG-BiP.ent and His-BiP.ent are about 2-fold higher than the activity exhibited by His-BiP and untagged recombinant murine BiP. After addition of pep2, both FLAG-BiP.ent and His-BiP reach the same k cat. This indicates that insertion of an enterokinase site between the N- and C-terminal domains increases the basal ATPase activity of BiP but does not have a profound effect on its structure or affinity for nucleotidic and peptidic substrates.Table IMichaelis and catalytic constants determined in steady state conditions for members of the HSP70 familyProteinK M(ATP)k catk catstimulated by pep2K M(pep2)Ref.μmminminμmFLAG-BiP.ent3 ± 10.57 ± 0.121 ± 0.5 (× 1.5–2.2)40This studyHis-BiP.ent4.0 ± 0.50.440.68 (× 1.5)200This studyHis-BiP1.9 ± 0.30.30 ± 0.150.9 ± 0.4 (× 2.6–3.4)100–120This studyRecombinant murine BiP0.40.14 ± 0.03NDND(13Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.-J.H. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar)Bovine Hsc700.6 ± 0.10.5 ± 0.3NDND(51Gao B. Eisenberg E. Greene L. Biochemistry. 1995; 34: 11882-11888Crossref PubMed Scopus (12) Google Scholar,56Benaroudj N. Fouchaq B. Ladjimi M.M. J. Biol. Chem. 1997; 272: 8744-8751Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar)Recombinant Hsc7024.70.86NDND(29Panzner S. Dreier L. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (323) Google Scholar)Hamster His-BiP1.50.4NDND(42Wei J. Hendershot L.M. J. Biol. Chem. 1995; 270: 26670-26676Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar)ATPase assays were carried out as follows: 20 μl of Binding Buffer containing 0.4 μm BiP and a final concentration of ATP equal to 100 μm (1 μCi of [γ-32P]ATP) were incubated 15 min at 37 °C with various concentrations of pep2 (0–3 mm). The reaction was stopped by addition of SDS (1% final), and the amount of inorganic 32P was quantified by phosphomolybdate extraction according to Seals et al. (59Seals J.M. McDonald J. Bruns D. Jarett L. Anal. Biochem. 1978; 90: 785-796Crossref PubMed Scopus (95) Google Scholar). ND, not determined. Open table in a new tab ATPase assays were carried out as follows: 20 μl of Binding Buffer containing 0.4 μm BiP and a final concentration of ATP equal to 100 μm (1 μCi of [γ-32P]ATP) were incubated 15 min at 37 °C with various concentrations of pep2 (0–3 mm). The reaction was stopped by addition of SDS (1% final), and the amount of inorganic 32P was quantified by phosphomolybdate extraction according to Seals et al. (59Seals J.M. McDonald J. Bruns D. Jarett L. Anal. Biochem. 1978; 90: 785-796Crossref PubMed Scopus (95) Google Scholar). ND, not determined. We investigated the degree of oligomerization of nucleotide-free FLAG-BiP.ent by size-exclusion chromatography at various protein concentrations (Fig. 1). FLAG-BiP.ent elutes in three major peaks. Peak A corresponds to aggregates that elute in the void volume. Peak O contains mostly trimeric and dimeric species. Peak M shoulders peak O and corresponds to monomeric species. The proportion of A, O, and M was found to vary slightly as a function of the concentration of FLAG-BiP.ent. At concentrations below 25 μm, the monomeric species are preponderant. By contrast, the amount of oligomers increases at higher concentration. The amount of aggregates remains constant over the range of concentrations investigated. To analyze the stability of monomeric and oligomeric species as a" @default.
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• W2077307831 title "Substrate Binding Induces Depolymerization of the C-terminal Peptide Binding Domain of Murine GRP78/BiP" @default.
• W2077307831 cites W1479915402 @default.
• W2077307831 cites W1520714027 @default.
• W2077307831 cites W1579202592 @default.
• W2077307831 cites W1597850573 @default.
• W2077307831 cites W17493816 @default.
• W2077307831 cites W1873199501 @default.
• W2077307831 cites W195389358 @default.
• W2077307831 cites W1964652518 @default.
• W2077307831 cites W1965261736 @default.
• W2077307831 cites W1965774536 @default.
• W2077307831 cites W1971800526 @default.
• W2077307831 cites W1978014671 @default.
• W2077307831 cites W1980772064 @default.
• W2077307831 cites W1985650639 @default.
• W2077307831 cites W1986732327 @default.
• W2077307831 cites W1988184759 @default.
• W2077307831 cites W1990671888 @default.
• W2077307831 cites W1996756446 @default.
• W2077307831 cites W1999157304 @default.
• W2077307831 cites W2011958996 @default.
• W2077307831 cites W2019858700 @default.
• W2077307831 cites W2026651883 @default.
• W2077307831 cites W2032536046 @default.
• W2077307831 cites W2034692794 @default.
• W2077307831 cites W2034943952 @default.
• W2077307831 cites W2035535257 @default.
• W2077307831 cites W2043693325 @default.
• W2077307831 cites W2043825401 @default.
• W2077307831 cites W2051636589 @default.
• W2077307831 cites W2056565816 @default.
• W2077307831 cites W2056816046 @default.
• W2077307831 cites W2060864422 @default.
• W2077307831 cites W2066744966 @default.
• W2077307831 cites W2070704594 @default.
• W2077307831 cites W2070809545 @default.
• W2077307831 cites W2076201930 @default.
• W2077307831 cites W2076945013 @default.
• W2077307831 cites W2086615828 @default.
• W2077307831 cites W2086922403 @default.
• W2077307831 cites W2087434802 @default.
• W2077307831 cites W2089457735 @default.
• W2077307831 cites W2089990328 @default.
• W2077307831 cites W2090425731 @default.
• W2077307831 cites W2092180405 @default.
• W2077307831 cites W2092198909 @default.
• W2077307831 cites W2092407683 @default.
• W2077307831 cites W2093527678 @default.
• W2077307831 cites W2099299706 @default.
• W2077307831 cites W2100837269 @default.
• W2077307831 cites W2101557398 @default.
• W2077307831 cites W2103778190 @default.
• W2077307831 cites W2113989206 @default.
• W2077307831 cites W2133924946 @default.
• W2077307831 cites W2143006558 @default.
• W2077307831 cites W4293247451 @default.
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