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• W1983077689 abstract "DnaJ, an Escherichia coli Hsp40 protein composed of 376 amino acid residues, is a chaperone with thioldisulfide oxidoreductase activity. We present here for the first time a small angle x-ray scattering study of intact DnaJ and a truncated version, DnaJ (1–330), in solution. The molecular weight of DnaJ and DnaJ (1–330) determined by both small angle x-ray scattering and size-exclusion chromatography provide direct evidence that DnaJ is a homodimer and DnaJ (1–330) is a monomer. The restored models show that DnaJ is a distorted ω-shaped dimeric molecule with the C terminus of each subunit forming the central part of the ω, whereas DnaJ (1–330) exists as a monomer. This indicates that the deletion of the C-terminal 46 residues of DnaJ impairs the association sites, although it does not cause significant conformational changes. Biochemical studies reveal that DnaJ (1–330), while fully retaining its thiol-disulfide oxidoreductase activity, is structurally less stable, and its peptide binding capacity is severely impaired relative to that of the intact molecule. Together, our results reveal that the C-terminal (331–376) residues are directly involved in dimerization, and the dimeric structure of DnaJ is necessary for its chaperone activity but not required for the thiol-disulfide oxidoreductase activity. DnaJ, an Escherichia coli Hsp40 protein composed of 376 amino acid residues, is a chaperone with thioldisulfide oxidoreductase activity. We present here for the first time a small angle x-ray scattering study of intact DnaJ and a truncated version, DnaJ (1–330), in solution. The molecular weight of DnaJ and DnaJ (1–330) determined by both small angle x-ray scattering and size-exclusion chromatography provide direct evidence that DnaJ is a homodimer and DnaJ (1–330) is a monomer. The restored models show that DnaJ is a distorted ω-shaped dimeric molecule with the C terminus of each subunit forming the central part of the ω, whereas DnaJ (1–330) exists as a monomer. This indicates that the deletion of the C-terminal 46 residues of DnaJ impairs the association sites, although it does not cause significant conformational changes. Biochemical studies reveal that DnaJ (1–330), while fully retaining its thiol-disulfide oxidoreductase activity, is structurally less stable, and its peptide binding capacity is severely impaired relative to that of the intact molecule. Together, our results reveal that the C-terminal (331–376) residues are directly involved in dimerization, and the dimeric structure of DnaJ is necessary for its chaperone activity but not required for the thiol-disulfide oxidoreductase activity. Hsp40 proteins collaborate specifically with Hsp70 proteins and some other factors to participate in a wide range of cellular processes essential to cell survival, such as assisting the folding, assembly, disassembly, and translocation of newly synthesized proteins, by suppressing aggregation or mediating degradation of misfolded proteins (1Georgopoulos C. Trends. Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 2Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar). The Hsp40 family proteins are divided into three subtypes (3Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (483) Google Scholar) (see Scheme 1). They all contain a J domain responsible for binding to the ATPase domain and stimulating the ATPase activity of Hsp70. Type I Hsp40 proteins, such as Escherichia coli DnaJ, yeast Yjd1, and human Hdj-2, have a Gly/Phe-rich region, which has been suggested to assist J domain interactions with Hsp70 (4Wall D. Zylicz M. Georgopoulos C. J. Biol. Chem. 1995; 270: 2139-2144Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The Gly/Phe-rich region is followed by a conserved cysteine-rich region forming a zinc finger domain and a poorly conserved C-terminal domain. The peptide binding site(s) has been found to locate within these two domains (5Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (274) Google Scholar, 6Banecki B. Liberek K. Wall D. Wawrzynow A. Georgopoulos C. Bertoli E. Tanfani F. Zylicz M. J. Biol. Chem. 1996; 271: 14840-14848Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 7Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Type II Hsp40 proteins, such as yeast Sis1 and mammalian Hdj-1, also contain the J, a Gly/Phe-rich region, and C-terminal domains but without the zinc finger domain (8Luke M. Sutton A. Arndt K.T. J. Cell Biol. 1991; 114: 623-638Crossref PubMed Scopus (145) Google Scholar, 9Zhong T. Arndt K.T. Cell. 1993; 73: 1175-1186Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Both type I and type II Hsp40 proteins act as molecular chaperones to bind and deliver nonnative proteins to Hsp70 (7Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 8Luke M. Sutton A. Arndt K.T. J. Cell Biol. 1991; 114: 623-638Crossref PubMed Scopus (145) Google Scholar, 10Caplan A.J. Douglas M.G. J. Cell Biol. 1991; 114: 609-621Crossref PubMed Scopus (211) Google Scholar), but they are not functionally equivalent (11Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Type III Hsp40 proteins, such as yeast YJL162c and virus T antigen, contain only the J domain, and they do not function as molecular chaperones (3Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (483) Google Scholar). In addition to the DnaK-dependent co-chaperone activity described above, DnaJ also exerts autonomous DnaK-independent chaperone activity (9Zhong T. Arndt K.T. Cell. 1993; 73: 1175-1186Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 12Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (787) Google Scholar, 13Linke K. Wolfram T. Bussemer J. Jakob U. J. Biol. Chem. 2003; 278: 44457-44466Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which is characterized by its ability to bind with unfolded proteins and prevent them from irreversible aggregation (10Caplan A.J. Douglas M.G. J. Cell Biol. 1991; 114: 609-621Crossref PubMed Scopus (211) Google Scholar, 11Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Moreover, DnaJ has been found to have thiol-disulfide oxidoreductase activity but little, if any, isomerase activity (14Tang W. Wang C.C. Biochemistry. 2001; 40: 14985-14994Crossref PubMed Scopus (29) Google Scholar). Although a large number of Hsp40 proteins have been discovered in prokaryotic and eukaryotic cells, only a few structures have been solved. These include the J domains of DnaJ (15Pellecchia M. Szyperski T. Wall D. Georgopoulos C. Wüthrich K. J. Mol. Biol. 1996; 260: 236-250Crossref PubMed Scopus (166) Google Scholar) and Hdj1 (16Qian Y.Q. Patel D. Hartl F.U. McColl D.J. J. Mol. Biol. 1996; 260: 224-235Crossref PubMed Scopus (138) Google Scholar), and the zinc finger domain of DnaJ (17Martinez-Yamout M. Legge G.B. Zhang O. Wright P.E. Dyson H.J. J. Mol. Biol. 2000; 300: 805-818Crossref PubMed Scopus (89) Google Scholar) solved by NMR 1The abbreviations used are: NMR, nuclear magnetic resonance; SAXS, small angle x-ray scattering; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DTT, dithiothreitol. 1The abbreviations used are: NMR, nuclear magnetic resonance; SAXS, small angle x-ray scattering; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DTT, dithiothreitol. methods, and the peptide-binding fragments of yeast Sis1 (18Sha B.D. Lee S. Cyr D.M. Struct. Fold. Des. 2000; 8: 799-807Abstract Full Text Full Text PDF Scopus (143) Google Scholar) and Ydj1 (19Li J. Qian X. Sha B. Structure (Camb.). 2003; 12: 1475-1483Abstract Full Text Full Text PDF Scopus (131) Google Scholar) solved by x-ray crystallographic techniques. To date, no structure has been reported for the entire Hsp40 molecule. DnaJ has been shown to exist as a homodimer of two 41 kDa-subunits, each composed of 376 amino acid residues, by size exclusion chromatography. Nevertheless, the structural elements responsible for dimerization have not been yet elucidated. Yeast type II Hsp40 protein, Sis1, has been shown to dimerize through a short C-terminal stretch of 15 amino acid residues, residues 338–352 (18Sha B.D. Lee S. Cyr D.M. Struct. Fold. Des. 2000; 8: 799-807Abstract Full Text Full Text PDF Scopus (143) Google Scholar). Considering that the C-terminal domain II (residues 260–337) of Sis1 is conserved between type I and II Hsp40 proteins, and the sequence (338–352) of Sis1 is highly homologous to residues 331–345 of DnaJ (8Luke M. Sutton A. Arndt K.T. J. Cell Biol. 1991; 114: 623-638Crossref PubMed Scopus (145) Google Scholar), we prepared a truncated DnaJ by removing the C-terminal residues 331–376. Using this truncated DnaJ (1–330) and full-length DnaJ, we investigated the structural and functional roles of residues 331–376 in maintaining the biological activities of DnaJ. Small angle x-ray scattering (SAXS) has recently been proven to be a powerful tool for investigating the solution structure of biomacromolecules. This, in part, is because of a significant improvement in the ab initio methods for restoring the three-dimensional shape of a molecule from the observed one-dimensional scattering profile in a model-independent manner (20Svergun D.I. Volkov V.V. Kozin M.B. Stuhrmann H.B. Acta Crystallogr. Sect. A. 1996; 52: 419-426Crossref PubMed Scopus (124) Google Scholar, 21Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1720) Google Scholar, 22Svergun D.I. Petoukhov M.V. Koch M.H.J. Biophys. J. 2001; 80: 2946-2953Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar, 23Chacon P. Moran F. Diaz J.F. Pantos E. Andreu J.M. Biophys. J. 1998; 74: 2760-2775Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), i.e. no detailed structural data, such as crystal structure coordinates, are required. In this communication we report, for the first time, determination of the low-resolution structure of full-length DnaJ and DnaJ (1–330) in solution using the SAXS technique with two independent ab initio shape-determination programs, DAMMIN (21Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1720) Google Scholar) and GASBOR (22Svergun D.I. Petoukhov M.V. Koch M.H.J. Biophys. J. 2001; 80: 2946-2953Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar). The results clearly indicate that the DnaJ molecule exists as a distorted ω-shaped homodimer with the C terminus of each subunit associated to form the central part of the ω, whereas the truncated DnaJ (1–330) fails to dimerize. Further, biochemical studies reveal that monomeric DnaJ (1–330) is severely deficient in peptide binding capacity and therefore lacks the autonomous chaperone activity. However, it retains the full thiol-disulfide oxidoreductase activity of the intact DnaJ homodimer. Proteins Expression and Purification—The expression plasmid pUHE21–2 containing the full-length E. coli DnaJ gene was generously donated by Dr. J. Buchner, Institut für Organische Chemie und Biochemie, TU München, Germany. DnaJ protein was expressed and purified according to Zylicz et al. (24Zylicz M. Yamamoto T. Mckittrick N. Sell S. Georgopoulos C. J. Biol. Chem. 1985; 260: 7591-7593Abstract Full Text PDF PubMed Google Scholar) and Tang and Wang (14Tang W. Wang C.C. Biochemistry. 2001; 40: 14985-14994Crossref PubMed Scopus (29) Google Scholar). The plasmid carrying the coding sequence for the M1-P330 fragment of DnaJ was constructed by cloning a DNA fragment with BamHI and PstI sites, amplified from the plasmid pUHE21-2, into the pBluscript II/SK(+) vector, and then subcloning into pUHE21-2 via the same restriction sites. Transformed XL1 blue cells were grown in 2 × YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl) containing 100 μg/ml ampicillin at 30 °C, and the overnight culture was diluted 100-fold and incubated for 6 h followed by induction for 4 h with 0.1 mm isopropyl 1-thio-β-d-galactoside. The cell pellet was suspended in buffer A (Tris buffer containing 50 mm NaCl) and sonicated. If not specified, 50 mm Tris-HCl buffer (pH 8.0) was used in all experiments and is referred to as Tris buffer. The supernatant was loaded onto a DEAE-Sepharose fast flow column equilibrated with buffer A. The flow-through out was then applied onto a Bio-Scale S5 column (Bio-Rad) equilibrated with buffer A and eluted with 10 ml and then 15 ml of Tris buffer containing 150 and 200 mm NaCl, respectively. Examination by SDS-PAGE showed that the fractions eluted with 200 mm NaCl were DnaJ (1–330) with a molecular mass of 36 kDa. The purified DnaJ and DnaJ (1–330) were stored in elution buffer at –80 °C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit muscle (25Liang S.J. Lin Y.Z. Zhou J.M. Tsou C.L. Wu P. Zhou Z. Biochim. Biophys. Acta. 1990; 1038: 240-246Crossref PubMed Scopus (70) Google Scholar) and E. coli DsbC, a periplasmic thiol-disulfide oxidoreductase (26Chen J. Song J.L. Zhang S. Wang Y. Cui D.F. Wang C.C. J. Biol. Chem. 1999; 274: 19601-19605Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), were kindly provided by G. P. Ren and Z. Zhao, respectively, of this group. Reduced and denatured RNase A was prepared essentially according to Pigiet and Schuster (27Pigiet V.P. Schuster B.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7643-7647Crossref PubMed Scopus (123) Google Scholar). Determinations—Concentrations of DnaJ and DnaJ (1–330) were determined by the Bradford method with bovine serum albumin (BSA) as a standard (28Bradford M.M. Anal. Biochem. 1976; 72: 248-255Crossref PubMed Scopus (214351) Google Scholar). Concentrations of BSA, GAPDH, DsbC, and native RNase A were determined spectrophotometrically at 280 nm with the absorption coefficients (A1 cm0.1%) of 0.66, 0.98, 0.7, and 0.695, respectively, and at 275 nm with ϵ275 nm = 9200 m for fully reduced RNase A (27Pigiet V.P. Schuster B.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7643-7647Crossref PubMed Scopus (123) Google Scholar). Homotetrameric GAPDH and homodimeric DnaJ were considered as protomers in the calculation of molar ratios. Intrinsic and ANS fluorescence spectra were measured in a Shimadzu RF-5301 PC spectrofluorometer at 25 °C with 280 nm and 380 nm for excitation, respectively. CD spectra in the far-ultraviolet region (200–250 nm) were determined using a Jasco 500 spectropolarimeter at 25 °C. SAXS Measurements—Synchrotron SAXS measurements of DnaJ and DnaJ (1–330) were carried out at the beamline BL-10C of Photon Factory, Tsukuba (29Ueki T. Hiragi Y. Kataoka M. Inoko Y. Amemiya Y. Izumi Y. Tagawa H. Muroga Y. Biophys. Chem. 1985; 23: 115-124Crossref PubMed Scopus (195) Google Scholar). The scattered signals were detected using a one-dimensional position-sensitive proportional counter with 512 channels. At a sample-to-detector distance of 1 m and an x-ray wavelength λ of 1.488 Å, the scattering vector q (q = 4π sinθ/λ, where 2θ is the scattering angle) ranged from 0.01 to 0.35 Å–1, which was calibrated using dried hen collagen as a standard oriented specimen. Samples were encapsulated inside a cell sandwiched by two thin parallel quartz windows 1 mm apart with a volume of 70 μl. All of the SAXS experiments were carried out in a sample holder maintained at 25.0 ± 0.1 °C. SAXS data of buffer and samples were collected alternatively in the frames of 60–300 s to avoid radiation-induced protein damage. Concentrations from 2.0 to 20.0 mg/ml for DnaJ and from 3.0 to 15.0 mg/ml for DnaJ (1–330) in Tris buffer containing 200 mm NaCl were used for the measurements of interparticle interactions. The data reduction includes normalization of the one-dimensional scattered data to the intensity of the transmitted beam and subtraction of the background scattering of the buffer. All of the scattering curves were then standardized to that of a protein concentration of 1 mg/ml. The low angle data were extrapolated to infinite dilution and merged with the high angle data measured at high protein concentrations to yield final scattering curves. Scattering Data Analysis—The scattering intensity I(q,c), expressed by the Guinier Equation (30Guinier A. Fournet G. Small Angle Scattering X-rays. John Wiley & Sons, New York1955Google Scholar), I(q,c)=I(0,c)exp(−Rg(c)2q23)(Eq. 1) is a function of scattering vector q and protein concentration c. Here I(0,c) is forward scattering intensity, and Rg(c) is apparent radius of gyration at finite concentration. At low protein concentrations, I(0,c) may be written by, Kc/I(0,c)=1/Mw+2A2c+…(Eq. 2) where Kc is a constant determined by using a series of concentrations of BSA as a reference protein with the known molecular mass of 67 kDa (31Porod G. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, London1982: 17-51Google Scholar). MW is the relative molecular weight of the protein and can be obtained by extrapolating Kc/I(0,c) to infinite dilution. A2 is the second virial coefficient resulting from interparticle interference effects and can discriminate between attractive and repulsive interactions. Repulsive interactions lead to positive values of A2, and attractive interactions lead to negative values. At the dilute limit, Rg(c) is given by, Rg(c)2=R02−Bifc+…(Eq. 3) where R0 is the radius of gyration at infinite dilution, and Bif is a parameter reflecting intersolute force potential (32Izumi Y. Kuwamoto S. Jinbo Y. Yoshino H. FEBS Lett. 2001; 495: 126-130Crossref PubMed Scopus (12) Google Scholar). The sign of Bif means the same as that of A2 (30Guinier A. Fournet G. Small Angle Scattering X-rays. John Wiley & Sons, New York1955Google Scholar). The three parameters A2, R0, and Bif were calculated using Equations 2 and 3. The pair-distance distribution function, P(r), given by, P(r)=12π2∫I(q)(qr)sin(qr)dq(Eq. 4) is a measure of the frequency of interatomic vector lengths within a protein molecule and can provide further information about the shape of the scattering particle. Ab Initio Molecular Shape Determination—The overall shapes of the proteins were restored from the experimental data by two independent programs, DAMMIN (21Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1720) Google Scholar) and GASBOR (22Svergun D.I. Petoukhov M.V. Koch M.H.J. Biophys. J. 2001; 80: 2946-2953Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar). Starting from a sphere with a diameter equal to the maximum particle size filled by ∼1000 densely packed small beads (dummy atoms), DAMMIN searches for the best dummy atom model through minimizing the discrepancy function, f(X) = χ2 + αP(X), between the calculated and experimental curves using the simulated annealing method. αP(X) is a looseness penalty with positive weight for α > 0, and χ is the discrepancy between the experimental Iexp(qj) and the calculated Icalc(qj) curves χ2=1N−1∑j[Iexp(qj)−cIcalc(qj)σ(qj)]2(Eq. 5) where N is the number of experimental points, c is a scaling factor, and σ(qj) is the experimental error at the momentum transfer qj. The method is to modify the coordinates of beads randomly, while always approaching the configurations that decrease the energy f(X). Starting from a random string, simulated annealing was employed to find a compact configuration of beads minimizing the discrepancy. In this method, a constant was subtracted from the experimental data to ensure that the intensity decay follows Porod's law for homogeneous particles. The x-ray scattering curves at high angles (q > 0.25 Å–1) contain a significant contribution from the internal particle structure. GASBOR is a more versatile ab initio approach, especially for high scattering vectors. A dummy residues model was generated by a random walk Cα chain and was folded to minimize the discrepancy between the calculated and the experimental scattering data. GASBOR uses a simulated annealing approach to find a chain-compatible spatial distribution of an exact number of dummy residues, which correspond to the Cα atoms of amino acid residues. The dummy residues method permits fitting data up to a resolution of 5 Å, if the number of amino acid residues of the protein is specified. Activity Assay—Thiol-disulfide reductase activity was assayed by measuring the turbidity increase at 650 nm because of insulin reduction (33Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar) and was expressed as a ratio of the slope of the linear part of the turbidity curve to the lag time (34Martínez-Galisteo E. Padilla C.A. Garcia-Alfonso C. López-Barea J. Barcena J.A. Biochimie. (Paris). 1993; 75: 803-809Crossref PubMed Scopus (42) Google Scholar). Oxidase activity was assayed by the oxidative reactivation of reduced and denatured RNase A (27Pigiet V.P. Schuster B.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7643-7647Crossref PubMed Scopus (123) Google Scholar). Aggregation during the refolding of fully denatured GAPDH upon dilution in the presence of DnaJ or DnaJ (1–330) was followed by light scattering according to Tang and Wang (14Tang W. Wang C.C. Biochemistry. 2001; 40: 14985-14994Crossref PubMed Scopus (29) Google Scholar) to measure the chaperone activity of the proteins. SAXS Parameters—As shown in Fig. 1 the Guinier plots of experimental SAXS data at all concentrations used were linear in the low q region, indicating that the proteins do not undergo aggregation. The function of Kc/I(0,c) evaluated from the intercepts of the Guinier plots is linear over the entire concentration range (Fig. 2A), and the slope represents the value of A2. The square of apparent radius of gyration Rg(c) calculated from the slopes of the Guinier plots, as a function of protein concentration, also showed a linear relation (Fig. 2B), and the slope represents the value of Bif (30Guinier A. Fournet G. Small Angle Scattering X-rays. John Wiley & Sons, New York1955Google Scholar). The SAXS parameters are summarized in Table I. The molecular mass of DnaJ, 94.7 kDa, is about twice the value 45.4 kDa for DnaJ (1–330). The values of A2 and Bif of DnaJ and DnaJ (1–330) are all negative, and the negative value of DnaJ (1–330) is ∼2–3-fold larger than that for DnaJ, indicating even stronger attractive interactions between the DnaJ (1–330) molecules. The radius of gyration of DnaJ, 62.1 ± 0.3 Å, is greater than the value 39.2 ± 0.3 Å for DnaJ (1–330).Table IStructural parameters of DnaJ and DnaJ (1–330) determined by SAXS experiments in solutionRg(0)aRadius of gyration at infinite dilution given by the Guinier approximation.RgbRadius of gyration at infinite dilution estimated using the GNOM package.MWcMolecular mass calculated from forward scattering intensities (Fig. 2A).A2dSecond virial coefficient calculated from Fig. 2A using Equation 2.BifeInterparticle interference coefficient calculated from Fig. 2B using Equation 3.ÅÅkDa10-4 ml mol g-210-13 cm5 g-1DnaJ62.1 ± 0.361.5 ± 0.394.7 ± 3.0-0.63 ± 0.2-23.5 ± 1.7DnaJ (1-330)39.2 ± 0.340.3 ± 0.345.4 ± 1.8-1.8 ± 0.3-46.5 ± 2.2a Radius of gyration at infinite dilution given by the Guinier approximation.b Radius of gyration at infinite dilution estimated using the GNOM package.c Molecular mass calculated from forward scattering intensities (Fig. 2A).d Second virial coefficient calculated from Fig. 2A using Equation 2.e Interparticle interference coefficient calculated from Fig. 2B using Equation 3. Open table in a new tab The pair-distance distribution function P(r) (Fig. 3) was obtained from the entire scattering curve I(q) in the range of q, from zero to qmax, by direct Fourier transformation (Equation 4). In the low angle region (q < 0.05 Å–1), I(q) was extended to zero by using the radius of gyration at infinite dilution, i.e. Rg (0) (Table I). In the high angle region (q > 0.05 Å–1), concentrated solutions were used to improve the statistics, as the scattering intensity is not affected by interparticle interactions. The maximum linear dimension, Dmax, determined from P(r) (Fig. 3) is 170 ± 5 Å for DnaJ and 120 ± 5 Å for DnaJ (1–330). The radius of gyration of DnaJ and DnaJ (1–330), estimated from the P(r) function by applying GNOM to the entire scattering profile in the range of 0.01 Å–1 to qmax, is 61.5 ± 0.3 Å and 40.3 ± 0.3 Å, respectively, which is consistent with the corresponding value estimated from the extrapolated Guinier plots. The P(r) functions calculated from the three-dimensional coordinates (PDB code 1BQ0) of the DnaJ sequence (1–77) (the J domain) and (PDB code 1EXK) of the sequence (131–209) (the zinc finger domain) using CRYSOL and GNOM were used to compare with those of DnaJ and DnaJ (1–330) (Fig. 3). The main peak at 47 Å and the Dmax of 120 Å for DnaJ (1–330) are both much larger than the corresponding values of 15 Å and 42 Å for the sequence 1–77 and 14 Å and 51 Å for the 131–209, respectively. The sum of the Dmax values for the sequences of 1–77 and 131–209, 93 Å, is smaller than the value of 120 Å obtained for DnaJ (1–330). This suggests that the two sequences and the fragment (210–330) are arranged to form an elongated DnaJ (1–330) molecule. DnaJ showed a spread P(r) profile with the main peak at 75 Å and a Dmax of 170 Å, which is, however, much smaller than twice the Dmax of DnaJ (1–330), indicating that the two DnaJ (1–330) subunits are not arranged linearly. Ab Initio Molecular Shape—Dummy atom modeling was performed from the scattering pattern up to qmax = 0.25 Å–1 using DAMMIN within a spherical search diameter of Dmax = 120 Å for DnaJ (1–330) and 170 Å for DnaJ without symmetrical constraint. The uniqueness and the stability of the restored shapes were checked by repeating modeling. A dozen independent models were then aligned and averaged using programs SUPCOMB (35Kozin M.B. Svergun D.I. J. Appl. Crystallogr. 2001; 34: 33-41Crossref Scopus (1080) Google Scholar) and DAMAVER (36Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1596) Google Scholar), which superimposed all models and determined the common envelope containing all models. In the next round, this common envelope was used as an initial search volume for all new models. Twelve runs of ab initio shape determination produced consistent results, as all output models yielded nearly identical scattering patterns (Fig. 4, dashed curves) with a discrepancy of χ = 1.3 for DnaJ (1–330) and χ = 1.1 for DnaJ to the respective corrected experimental data obtained by subtracting a constant in the scattering range up to 0.25 Å–1. These final models were again aligned and averaged using SUPCOMB (35Kozin M.B. Svergun D.I. J. Appl. Crystallogr. 2001; 34: 33-41Crossref Scopus (1080) Google Scholar) and DAMAVER (36Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1596) Google Scholar). The averaged dummy atom model of DnaJ at three orthogonal orientations (Fig. 5, left column) shows a molecule basically with a 2-fold symmetry along the middle line of the front view.Fig. 5Restored models of DnaJ.A, front view; B, side view; C, top view. Left column, the averaged model by DAMMIN using a qmax of 0.25 Å–1 with no symmetrical constraint. Right column, the most probable model (dummy residues) by GASBOR using a qmax of 0.35 Å–1 with a P2 symmetry.View Large Image Figure ViewerDownload (PPT) The restored dummy residues models were obtained by performing 12 runs with the GASBOR program using 752 dummy residues assuming no molecular symmetry and fitting the scattering profiles up to qmax = 0.35 Å–1, i.e. with a resolution of 2π /qmax = 18 Å. The results are similar to those obtained by DAMMIN (data not shown). The models restored by both DAMMIN and GASBOR exhibit 2-fold symmetry. A 2-fold (P2) symmetrical constraint in GASBOR was employed using all 376 residues for one DnaJ monomer to obtain more reliable models. The final models fit the experimental data very well with a discrepancy of χ = 1.3 in the entire scattering range as indicated by the solid curves in Fig. 4. DAMSEL in the package of DAMAVER was employed to align all of the possible pairs of models and to identify the most probable model, which should yield the smallest average discrepancy (Fig. 5, right column). The results suggest that DnaJ exhibits an ω-shape. Using the same approach described above, DnaJ (1–330) was restored to appear as an elongated roughly S-shaped molecule composed of three regions (Fig. 6, left column). The NMR structures of fragments 1–77 and 131–209 were accommodated well within the envelope of the restored model of DnaJ (1–330). The NMR structure of the zinc finger domain (fragment 131–209) was superimposed with the restored DnaJ (1–330) model next to the J domain. The zinc finger domain is composed of two zinc fingers in an abnormal topology (17Martinez-Yamout M. Legge G.B. Zhang O. Wright P.E. Dyson H.J. J. Mol. Biol. 2000; 300: 805-818Crossref PubMed Scopus (89) Google Scholar). It has been reported that zinc finger 1 is necessary for the autonomous chaperone activity in cooperation with the C-terminal domain (5Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (274) Google Scholar, 7Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 1" @default.
• W1983077689 created "2016-06-24" @default.
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