Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m203398200
ISSN1083-351X
AutoresAlison E. Ashcroft, Achim Brinker, Joseph E. Coyle, Frank Weber, Markus Kaiser, Luis Moroder, Mark R. Parsons, Joachim Jäger, Ulrich Hartl, Manajit Hayer‐Hartl, Sheena E. Radford,
Tópico(s)Enzyme Structure and Function
ResumoAdvances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer. Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer. GroEL (residues 191–376) ApEL containing the mutations G337S and I349E helix D mobile loop GroEL (residues 188–381) with a C-terminal hexahistidine tag GroEL (residues 191–376) with an N-terminal hexahistidine tag strong binding peptide with a C-terminal maleimide intact GroEL containing the mutation N229C and in which all the endogenous cysteine residues have been exchanged for alanine intact GroEL containing the mutation N229C and in which all the endogenous cysteine residues have been exchanged for alanine, with the 229C covalently bound to SBP-Mal GroES mutant in which a cysteine residue has been added to the C terminus wild-type GroEL N-(9-fluorenyl)methoxycarbonyl dithiothreitol 5-dimethylaminonaphthalene-1-sulfonyl 4-morpholinepropanesulfonic acid root mean square Several classes of molecular chaperones assist in the folding of newly synthesized polypeptides by preventing off-pathway reactions that lead to aggregation (1Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar, 2Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2429) Google Scholar, 3Hartl F.U. Hayer-Hartl M.K. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2792) Google Scholar). The so-called chaperonins (4Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulos C.P. Hendrix R.W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (933) Google Scholar) are large cylindrical structures that transiently enclose a partially folded polypeptide and allow it to continue folding in a sequestered environment, blocking intermolecular associations between chains during folding. The chaperonin of Escherichia coli, GroEL, belongs to the group I class of chaperonins and has been studied in great detail (e.g. Refs. 5Grallert H. Buchner J. J. Struct. Biol. 2001; 135: 95-103Crossref PubMed Scopus (71) Google Scholar and 6Kusmierczyk A.R. Martin J. Mol. Biotechnol. 2001; 19: 141-152Crossref PubMed Scopus (7) Google Scholar). GroEL interacts with an estimated 10–15% of newly synthesized polypeptides in the bacterial cytosol (7Ewalt K.L. Hendrick J.P. Houry W.A. Hartl F.U. Cell. 1997; 90: 491-500Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 8Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (435) Google Scholar). GroEL is a homotetradecamer of ∼57-kDa subunits that are arranged as two heptameric rings stacked back-to-back (9Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1194) Google Scholar, 10Roseman A.M. Chen S.X. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). GroEL requires the presence of a co-chaperone, GroES, together with a controlled cycle of nucleotide binding and hydrolysis, to complete its functional cycle (1Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar,11Feltham J.L. Gierasch L.M. Cell. 2000; 100: 193-196Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 12Sigler P.B., Xu, Z. Rye H.S. Burston S.G. Fenton W.A. Horwich A.L. Annu. Rev. Biochem. 1998; 67: 581-608Crossref PubMed Scopus (477) Google Scholar). GroES is a single heptameric ring of identical ∼10-kDa subunits that binds in a nucleotide-dependent manner to one end of the GroEL cylinder. A substrate protein binds with highest affinity to the nucleotide-free state of GroEL (reviewed in Ref. 1Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar). On subsequent binding of 7 ATP and GroES, the substrate protein is jettisoned into a cis-folding cage where it is free to fold to its final native form. Whereas the nucleotide-coupled interplay between GroEL, GroES, and protein substrate is understood in great detail, precisely how GroEL binds a wide array of non-native proteins and assists their folding remains elusive. Each GroEL subunit consists of an equatorial, an apical, and an intermediate domain (9Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1194) Google Scholar, 10Roseman A.M. Chen S.X. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). The equatorial domains of the subunits contain the ATP-binding site and both the N and C termini; they also mediate most inter-subunit contacts within and between rings. The apical domains form the entrance to the GroEL cavity and contain the residues involved in protein and GroES binding (13Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (574) Google Scholar). Each intermediate domain forms a hinge-like connector between the equatorial and apical domains of that subunit. Studies of polypeptide recognition by GroEL using various techniques (reviewed in Ref. 14Coyle J.E. Jaeger J. Grob M. Robinson C.V. Radford S.E. Fold and Des. 1997; 2: R93-R104Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) suggest that the binding site involves the apical domains; more recent crystallographic studies suggest that helices H and I within the apical domain form the substrate-binding site (15Smote A.L. Panda M. Brazil B.T. Buckle A.M. Fersht A.R. Horowitz P.M. Biochemistry. 2001; 40: 4484-4492Crossref PubMed Scopus (66) Google Scholar, 16Wang Q.H. Buckle A.M. Fersht A.R. J. Mol. Biol. 2000; 304: 873-881Crossref PubMed Scopus (20) Google Scholar, 17Chatellier J. Hill F. Fersht A.R. J. Mol. Biol. 2000; 304: 883-896Crossref PubMed Scopus (16) Google Scholar, 18Chatellier J. Hill F. Foster N.W. Goloubinoff P. Fersht A.R. J. Mol. Biol. 2000; 304: 897-910Crossref PubMed Scopus (28) Google Scholar, 19Chen L.L. Sigler P.B. Cell. 1999; 99: 757-768Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 20Ma J.P. Sigler P.B., Xu, Z.H. Karplus M. J. Mol. Biol. 2000; 302: 303-313Crossref PubMed Scopus (205) Google Scholar). Interestingly, these helices are the least well defined regions in the GroEL crystal structure and have relatively high thermal parameters (B-factors) even in the isolated apical domain (21Braig K. Adams P.D. Brunger A.T. Nat. Struct. Biol. 1995; 2: 1083-1094Crossref PubMed Scopus (230) Google Scholar, 22Buckle A.M. Zahn R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3571-3575Crossref PubMed Scopus (201) Google Scholar), raising the possibility that the ability of GroEL to bind a diverse range of polypeptides may involve this conformational plasticity. Structural mobility and exposed hydrophobic surfaces are features common to both the apical domains of GroEL and non-native polypeptides. Here we investigate the properties of the apical domain from GroEL and its peptide binding characteristics using a range of analytical techniques. Specifically, the ability of electrospray mass spectrometry (ESI-MS) to simultaneously observe different protein conformations in solution was exploited to study the conformational dynamics of the GroEL apical domain and the ability of different conformers to bind peptide mimics of the co-chaperone GroES and the substrate protein, rhodanese. Parallel fluorescence emission studies were used to study these complexes and compare the binding properties of the apical domain constructs for each peptide with those of intact GroEL. The data suggest that the apical domain possesses different binding sites for the peptides in that no competition for binding individual peptides to the apical domain was observed. Finally, using surface plasmon resonance and fluorescence studies on a GroEL variant in which the GroES-binding groove is blocked by a covalently bound peptide, we show that the substrate and the GroEL mobile loop-binding sites are at least partially distinct even in the intact GroEL tetradecamer. The apical domain constructs: ApEL1(GroEL-(191–376)), N-His ApEL (GroEL-(191–376) containing an N-terminal hexahistidine tag), C-His ApEL (GroEL-(188–381) containing a C-terminal hexahistidine tag), and ApTrap (ApEL G337S/I349E) were cloned, expressed, and purified as described previously (23Weber F. Keppel F. Georgopoulos C. Hayer-Hartl M.K. Hartl F.U. Nat. Struct. Biol. 1998; 5: 977-985Crossref PubMed Scopus (69) Google Scholar, 24Weber F. Keppel F. Georgopoulos C. Hayer-Hartl M.K. Hartl F.U. Nat. Struct. Biol. 1999; 6: 200-201Crossref Scopus (2) Google Scholar). The concentration of all apical domain constructs was determined using an extinction coefficient of 4260 m−1cm−1 at 280 nm, calculated by the method of Pace et al. (25Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3452) Google Scholar). Wild-type GroEL and EL N229C (intact GroEL containing the mutation N229C and in which all the endogenous cysteine residues have been exchanged for alanine) were purified as described previously (26Brinker A. Pfeifer G. Kerner M.J. Naylor D.J. Hartl F.U. Hayer-Hartl M. Cell. 2001; 107: 223-233Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). All crystallization screens were performed by vapor diffusion using the hanging drop method at 18 °C. Sparse matrix screens were used to perform initial crystallization trials in 24-well plates with a well volume of 500 μl (27Jancarik J. Kim S.H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2079) Google Scholar). Lyophilized C-His ApEL was dissolved in, and extensively dialyzed against, buffer (20 mm Tris-HCl, 1 mm DTT, pH 7.2) prior to screening. The final protein solution was 10 mg ml−1. Hanging drops contained equal volumes (2 μl each) of the protein and well solutions. Crystals of C-His ApEL were obtained after 1–2 weeks with a well solution of 0.6 m NaCl, 14% (w/v) PEG 6000, 100 mm Tris-HCl, pH 8.5. Crystals of C-His ApEL were transferred to a solution of mother liquor containing 15% (v/v) glycerol as a cryoprotectant and rapidly frozen in liquid nitrogen prior to data collection. X-ray data were collected using a 30-cm MAR Research image plate detector at the Synchrotron Radiation Source Station 9.6 (Daresbury, UK) (λ = 0.87 Å). Indexing and extraction of the raw x-ray intensities were performed using the program MOSFLM (28Leslie A.G.W. Joint CCP4 and ESF-EACMB Newsletter Protein Crystallography. 26. Daresbury Laboratory, Warrington, UK1992Google Scholar). Intensities were merged and amplitudes were calculated using programs from the CCP4 program suite (29Collaborative Computational Project, N. Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Molecular replacement was carried out with the program AMORE (30Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar) using an initial search model consisting of residues 188–381 from the refined GroEL tetradecamer (21Braig K. Adams P.D. Brunger A.T. Nat. Struct. Biol. 1995; 2: 1083-1094Crossref PubMed Scopus (230) Google Scholar). The high resolution structure of an apical domain fragment closely resembling our construct, containing GroEL residues 191–376 and an N-terminal histidine tag sequence (22Buckle A.M. Zahn R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3571-3575Crossref PubMed Scopus (201) Google Scholar), was used subsequently as a model for further modeling and refinement. Initial model building was carried out with the program FRODO (31Pflugrath J.W. Saper M.A. Quiocho F.A. Hall S. Ashira T. New Generation Graphics System for Molecular Modelling, Methods and Applications in Crystallographic Computing. Claredon Press, Oxford1984Google Scholar, 32Jones T.A. J. Appl. Crystallogr. 1978; 15: 268-272Crossref Google Scholar) and the structure was refined first at medium resolution using X-PLOR (33Brunger A.T. XPLOR: A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1993Google Scholar). Inspection of the resulting model using the program O (34Jones T.A. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) and full refinement at high resolution including all data was performed using the programs CNS (35Adams P.D. Pannu N.S. Read R.J. Brunger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar) and REFMAC (36Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar). The peptides helix D (HD) of rhodanese (amino acid residues 248–267) (37Ploegman J.H. Drent G. Kalk K.H. Hol W.G. Heinrikson R.L. Keim P. Weng L. Russell J. Nature. 1978; 273: 245-249Crossref PubMed Scopus (214) Google Scholar) (HD: Ac-RKGVTACHIALAAYLCGKPD-NH2) and mobile loop (ML) of GroES (amino acid residues 13–32) (ML: Ac-KRKEVETKSAGGIVLTGSAA-NH2) were chemically synthesized with a C-terminal amide on an automated peptide synthesizer (Applied Biosystems, Foster City, CA, model 430A) using standard Fmoc methodology and purified by reverse phase high performance liquid chromatography. The N terminus was modified with either an acetyl or dansyl group, the latter to provide a sensitive fluorescent probe for monitoring binding to the apical domain. The purity and molecular masses of the peptides were verified by analysis of a 10 ng μl−1 solution in 1:1 (v/v) acetonitrile, 0.05% aqueous formic acid using positive ionization ESI-MS. The concentrations of dansylated peptides were determined using the extinction coefficient for a single dansyl group of 4500m−1 cm−1 at 330 nm (38Hutchinson J.P. Oldham T.C. Elthaher T.S.H. Miller A.D. J. Chem. Soc. Perkin Trans. 1997; 2: 279-288Crossref Scopus (22) Google Scholar). The concentrations of non-dansylated peptides were estimated by dry weight. The apical domain constructs were analyzed at a concentration of 20 μm by continuous infusion at a flow rate of 5 μl min−1 into the electrospray ionization source of a Platform II (Micromass UK Ltd., Manchester, United Kingdom) single quadrupole mass spectrometer using a syringe pump (model 22, Harvard Apparatus, Holliston, MA). Where "native" electrospray conditions were used, the samples were dissolved in ammonium acetate (50 mm) at pH 7.5. Where "denaturing" electrospray conditions were used, the samples were analyzed in 1:1 (v/v) 0.1% aqueous formic acid, acetonitrile. In both cases the ionization source was maintained at 30 °C and nitrogen was employed as both the nebulizing and drying gases at flow rates of 20 and 200 l h−1, respectively. Positive ionization electrospray was used with a capillary voltage of 2.3 kV, the counter electrode set at 0 kV, and the sampling cone at 30 V. Data were acquired over the range m/z 500–3000 at a scan speed of 10 s and processed using MassLynx software (Micromass UK Ltd.). An external calibration using horse heart myoglobin (molecular mass 16,951.5 Da; Sigma) was applied to ensure mass accuracy. The m/z spectra displayed were smoothed mildly using a Sovitzy-Golay algorithm. The molecular mass (zero charge state) profiles illustrated were generated from the m/zspectra using the Maximum Entropy (39Ferrige A.G. Seddon M.J. Green B.N. Jarvis S.A. Skilling J. Rapid Commun. Mass Spectrom. 1992; 6: 707-711Crossref Scopus (241) Google Scholar) software supplied with MassLynx. To determine the thermal stability of the apical domain constructs, the ionization source temperature was raised in a stepwise fashion from 30–190 °C. The apical domain was mixed with the peptides HD or ML in ammonium acetate (50 mm) at pH 7.5 and the mixtures were incubated for 1 h at ambient temperature prior to ESI-MS analysis. The concentration of the apical domain was maintained at a constant 20 μm although the concentration of ML was varied from 1 to 200 μm and the concentration of HD was maintained at 50 μm to maximize the proportion of the complex without incurring problems with aggregation. The ionization source temperature was maintained at 30 °C and the sampling cone voltage at 30 V; these conditions were chosen to preserve protein-peptide complexation. N-Hydroxysuccinimido maleoyl-β-alaninate (Mal>β-Ala-OSu) was prepared following a previously published procedure (40Romano R. Bayerl T.M. Moroder L. Biochim. Biophys. Acta. 1993; 1151: 111-119Crossref PubMed Scopus (20) Google Scholar). The precursor peptide Ac-SWMTTPWGFLHP (the so-called strong-binding peptide, SBP (19Chen L.L. Sigler P.B. Cell. 1999; 99: 757-768Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar)) was synthesized with an automated peptide synthesizer (Applied Biosystems model 430A) using standard Fmoc/tBu chemistry on pre-loaded Fmoc-Lys(Boc)-2-chlorotritylresin (loading: 0.62 mmol g−1, 194 mg, 0.12 mmol). After cleavage and deprotection of the peptide with 95:2:3 (v/v/v) trifluoroacetic acid:water:triisopropyl silane and precipitation with methyl t-butylether, the precipitate was dissolved in 4:1 (v/v) t-butanol:water and lyophilized to produce a yield of 162 mg. The crude product (62 mg) was dissolved inN, N-dimethylformamide (20 ml) and reacted overnight with Mal>β-Ala-OSu (10.5 mg) and diisopropylethylamine (20.3 μl). After evaporation, the residue was dissolved in 4:1 (v/v) t-butanol:water and lyophilized. The resulting product was purified on a NucleosilTM C18 column using a linear gradient of 30–80% (v/v) aqueous acetonitrile in 60 min. The product-containing fractions were pooled and the solution was dried. The residue was dissolved in 4:1 (v/v) t-butanol:water and lyophilized to yield 12 mg of 98% pure material as verified by high performance liquid chromatography and mass spectrometric analysis. A ∼3-fold molar excess of SBP-Mal over EL N229C cysteines was applied to achieve quantitative modification. SBP-Mal (10 mm in Me2SO) was diluted 20-fold with H2O. 200 μl of EL N229C (to give a final concentration 9 μm) was added to 1 ml of 0.5 mm peptide solution and incubated for 1 h at 25 °C. The reaction was stopped by the addition of β-mercaptoethanol (50 mm) and excess reagent was removed by gel filtration chromatography. Less than 10% free thiols were detected in EL N229C:SBP-Mal on treatment with maleimidosalicylic acid. Denatured rhodanese (100 μm in 6 m guanidinium chloride) was diluted 200-fold into buffer (20 mm MOPS, pH 7.4, 100 mm KCl, 5 mm MgCl2) in the absence or presence of 1.0 μm EL N229C or EL N229C:SBP-Mal. Rhodanese aggregation was followed by turbidity measurements at 320 nm. Steady state fluorescence emission was measured at 20 °C on a spectrofluorimeter (model LS50B, PerkinElmer Life Sciences) using a 1-cm path length. Protein-peptide complexes were equilibrated for 5 min prior to acquiring fluorescence emission spectra. Fluorescence emission scans of dansylated peptides were recorded between 450 and 600 nm, using an excitation wavelength of 350 nm and a scan speed of 60 nm min−1. Excitation and emission slit widths were typically between 5 and 10 nm. Peptide binding titrations were performed with similar parameters but in the time drive mode, measuring emission at a single wavelength. The emission wavelength used was 500 nm for HD and 535 nm for ML binding to ApEL, respectively. All titrations were performed by adding small volumes of the dansyl-ML or ApEL to a cuvette containing 2–3 ml of either ApEL (25 mm) or dansyl-HD (3 mm), respectively, in buffer (50 mm Hepes, pH 7.5, 2 mm DTT). After each addition, the solution was mixed thoroughly and allowed to equilibrate thermally for ∼5 min. Titration of ApEL with the dansyl-ML required ∼25-min equilibration times. The total volume of ligand added never exceeded 10% of the total volume. For each assay, a control experiment was performed by adding the ligand to a solution of buffer alone and was subtracted from the corresponding reading acquired in the presence of protein or peptide. All peptide binding curves were fitted using the program GrafitTM (Erithacus Software Ltd.). Single transition ligand-binding profiles were fitted with either a weak (Equation 1) or tight (Equation 2) ligand binding equation as appropriate (41Ranson N.A. Burston S.G. Clarke A.R. J. Mol. Biol. 1997; 266: 656-664Crossref PubMed Scopus (81) Google Scholar). Typically, the weak binding equation was used under conditions where the dissociation constant was greater than the concentration of binding sites and the tight ligand binding equation was used under conditions where the concentration of binding sites was much greater than the dissociation constant. F=(Fmax[L])Kd+[L]Equation 1 Where F is the signal, Fmax is the maximum signal, Kd is the dissociation constant, and L is the concentration of ligand. F=(E0+kd+L)+((E0+kd+L)2-(4EoL)1/2)2Eo×[Fmax-Fmin]+FminEquation 2 Where F is the signal, Fmaxand Fmin are the maximum and minimum signals, respectively, E0 is the protein ligand-binding site concentration, Kd is the dissociation constant, and L is the total ligand concentration. Mutant GroES 98C (42Rye H.S. Roseman A.M. Chen S.X. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) (mutant co-chaperone GroES with an additional cysteine at the C-terminal) was purified as described previously (26Brinker A. Pfeifer G. Kerner M.J. Naylor D.J. Hartl F.U. Hayer-Hartl M. Cell. 2001; 107: 223-233Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) and immobilized (300 resonance units) via a thioether linkage on a CM5 biosensor chip (Biacore 2000 SPR instrument, Biacore AB, Uppsala, Sweden) and the analysis was performed as described previously (43HayerHartl M.K. Martin J. Hartl F.U. Science. 1995; 269: 836-841Crossref PubMed Scopus (138) Google Scholar). Binding was followed using buffer (20 mm MOPS, pH 7.4, 100 mm KCl, 5 nm MgCl2, 2 mm ATP) at a flow rate of 20 μl min−1 at 25 °C. The concentration of GroEL was 250 nm. Various GroEL apical domain fragments, mainly spanning amino acids 191–376 (with or without a hexahistidine tag) were expressed and purified for these studies (Fig.1A). ApTrap contains two point mutations in GroEL-(191–376): G337S and I349E, which in intact wild-type (WT) GroEL result in loss of ability of GroEL to release bound polypeptide (13Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (574) Google Scholar, 44Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (331) Google Scholar). Additionally, C-His ApEL is a slightly larger apical domain construct, encompassing residues GroEL-(188–381), containing a C-terminal hexahistidine tag. Several crystal structures of GroEL apical domains have been obtained to date (9Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1194) Google Scholar, 19Chen L.L. Sigler P.B. Cell. 1999; 99: 757-768Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 22Buckle A.M. Zahn R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3571-3575Crossref PubMed Scopus (201) Google Scholar, 45Boisvert D.C. Wang J. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (245) Google Scholar, 46Zahn R. Buckle A.M. Perret S. Johnson C.M. Corrales F.J. Golbik R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15024-15029Crossref PubMed Scopus (136) Google Scholar). To compare the structure of these domains with that of the larger construct C-His ApEL constructed here, crystals of C-His ApEL were grown as described under "Experimental Procedures." Crystals appeared within 1–2 weeks and belong to space groupP212121 with unit cell dimensions of a = 48.6 Å, b = 61.9 Å, and c = 75.2 Å. A 98.8% complete data set was collected at cryogenic temperatures from a single crystal to 2.06-Å resolution. The molecular replacement procedure revealed a clear solution in both the rotation and translation searches. The correlation coefficient corresponding to the highest peak in the translation function was 0.600 and the initial R-factor after rigid-body optimization was 0.387 (47Brunger A.T. Kurijan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar). The crystal structure of the apical domain fragment, C-His ApEL (residues 188–381), is well defined. The free R of the final refined model is 0.257 including 95% of all data between 20 and 2.06 Å. In the current model the deviations in ideal bond lengths and bond angles were 0.011 Å and 2.4°, respectively, and the average real-space correlation coefficient for all amino acid residues was 0.936. The backbone torsion angles of all residues are within the allowed regions of the Ramachandran plot (data not shown). The overall data collection and model refinement statistics are collated in TableI and the coordinates have been deposited in the Protein Data Bank as 1LA1.Table ICrystallographic statistics for the apical domain construct C-His ApELCrystallographic statisticsModel C-His ApEL− (188–381)Unit cell dimensionsa = 48.6 Å, b = 61.9 Å, c = 75.2 ÅSpace groupP212121Number of reflections (obs./poss.)14319/15933Overall completeness98.8%Significance 〈I/ςIΣ10.1Resolution limits20.0–2.06 ÅNo. of non-hydrogen atoms1448No. of solvent molecules447CrystallographicR-factor0.197Free R-factor0.257R.m.s bond0.011 ÅR.m.s angle2.4 °R.m.s. dihedrals23.90 ° Open table in a new tab C-His ApEL shares a virtually identical fold to its counterparts in both the intact GroEL tetradecamer as well as in the shorter domains 191–336, 191–345, 193–345, and 191–376 (9Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1194) Google Scholar, 16Wang Q.H. Buckle A.M. Fersht A.R. J. Mol. Biol. 2000; 304: 873-881Crossref PubMed Scopus (20) Google Scholar, 19Chen L.L. Sigler P.B. Cell. 1999; 99: 757-768Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 22Buckle A.M. Zahn R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3571-3575Crossref PubMed Scopus (201) Google Scholar, 46Zahn R. Buckle A.M. Perret S. Johnson C.M. Corrales F.J. Golbik R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15024-15029Crossref PubMed Scopus (136) Google Scholar). A comparison of five independent models of the apical domain of GroEL, namely C-His ApEL (determined here), N-His ApEL (22Buckle A.M. Zahn R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3571-3575Crossref PubMed Scopus (201) Google Scholar), ApEL-(191–336)·peptide complex (19Chen L.L. Sigler P.B. Cell. 1999; 99: 757-768Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), intact GroEL (9Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1194) Google Scholar), and a GroEL·ADP complex (45Boisvert D.C. Wang J. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. S
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