Solution Structure of the Tandem Src Homology 3 Domains of p47 in an Autoinhibited Form
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m401457200
ISSN1083-351X
AutoresSatoru Yuzawa, Kenji Ogura, Masataka Horiuchi, Nobuo Suzuki, Yūko Fujioka, Mikio Kataoka, Hideki Sumimoto, Fuyuhiko Inagaki,
Tópico(s)Adenosine and Purinergic Signaling
ResumoThe phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O2.) that kill invading microorganisms. p47phox is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47phox tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22phox. The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O2.) that kill invading microorganisms. p47phox is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47phox tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22phox. The NADPH oxidase catalyzes the reduction of oxygen to a superoxide anion using NADPH as an electron donor, which plays a critical role to kill invading microorganisms in neutrophils and other phagocytic cells. The phagocyte NADPH oxidase comprises membrane-bound flavocytochrome b558, a heterodimer of gp91phox and p22phox, and at least four cytosolic regulatory subunits consisting of p47phox, p67phox, p40phox, and Rac (1Nauseef W.M. Proc. Assoc. Am. Phys. 1999; 111: 373-382Crossref PubMed Scopus (85) Google Scholar, 2Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 3Segal B.H. Leto T.L. Gallin J.I. Malech H.L. Holland S.M. Medicine. 2000; 79: 170-200Crossref PubMed Scopus (718) Google Scholar, 4Lambeth D.J. J. Biochem. Mol. Biol. 2000; 33: 427-439Google Scholar, 5Takeya R. Sumimoto H. Mol. Cells. 2003; 16: 271-277PubMed Google Scholar). Upon activation of the cell, the p47phox-p67phox-p40phox complex translocates from the cytosol to the plasma membrane to associate with flavocytochrome b558, so that the NADPH oxidase subunits are assembled into the activated complex. The interaction between p47phox and p22phox is considered to be essential for NADPH oxidase activation (6Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (254) Google Scholar, 7Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (240) Google Scholar, 8Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The p47phox subunit contains a phox homology domain, tandem SH3 domains, a polybasic region/autoinhibitory region (PBR/AIR), 1The abbreviations used are: SH3 domain, Src homology domain 3; PBR, polybasic region; AIR, autoinhibitory region; SAXS, small angle x-ray scattering; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser enhancement; RDC, residual dipolar coupling; IP, in-phase; AP, anti-phase; N-SH3, the N-terminal SH3 domain; C-SH3, C-terminal SH3 domain.1The abbreviations used are: SH3 domain, Src homology domain 3; PBR, polybasic region; AIR, autoinhibitory region; SAXS, small angle x-ray scattering; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser enhancement; RDC, residual dipolar coupling; IP, in-phase; AP, anti-phase; N-SH3, the N-terminal SH3 domain; C-SH3, C-terminal SH3 domain. and a proline-rich region in this order (Fig. 1A). In resting cells, the tandem SH3 domains of p47phox are masked through an intramolecular interaction with PBR/AIR, resulting in an autoinhibited form (6Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (254) Google Scholar, 7Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (240) Google Scholar, 9de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (84) Google Scholar, 10Ago T. Nunoi H. Ito T. Sumimoto H. J. Biol. Chem. 1999; 274: 33644-33653Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 11Huang J. Kleinberg M.E. J. Biol. Chem. 1999; 274: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Upon cell stimulation, a number of serine residues in PBR/AIR are phosphorylated (12El Benna J. Faust L.P. Babior B.M. J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar, 13El Benna J. Faust L.P. Johnson J.L. Babior B.M. J. Biol. Chem. 1996; 271: 6374-6378Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 14Fontayne A. Dang P.M. Gougerot-Pocidalo M.A. El Benna J. Biochemistry. 2002; 41: 7743-7750Crossref PubMed Scopus (326) Google Scholar, 15Hoyal C.R. Gutierrez A. Young B.M. Catz S.D. Lin J.H. Tsichlis P.N. Babior B.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5130-5135Crossref PubMed Scopus (150) Google Scholar). Phosphorylation of p47phox induces conformational changes that subsequently lead to rearrangements in intramolecular interactions and the exposure of the tandem SH3 domains that enable interaction with the p22phox subunit in flavocytochrome b558 (6Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (254) Google Scholar, 10Ago T. Nunoi H. Ito T. Sumimoto H. J. Biol. Chem. 1999; 274: 33644-33653Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 11Huang J. Kleinberg M.E. J. Biol. Chem. 1999; 274: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Anionic amphiphiles such as arachidonate and SDS, activators of the NADPH oxidase in vitro, facilitate a conformational change of p47phox, exposing its SH3 domains as well as inducing phosphorylation (6Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (254) Google Scholar, 16Swain S.D. Helgerson S.L. Davis A.R. Nelson L.K. Quinn M.T. J. Biol. Chem. 1997; 272: 29502-29510Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Shiose A. Sumimoto H. J. Biol. Chem. 2000; 275: 13793-13801Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Recently, the structures of the autoinhibited and the activated forms of the tandem SH3 domains of p47phox (Protein Data Bank codes 1NG2 and 1OV3) were reported (18Groemping Y. Lapouge K. Smerdon S.J. Rittinger K. Cell. 2003; 113: 343-355Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). We also determined the structure of the tandem SH3 domains in the autoinhibited form (p47phox(151-340); Protein Data Bank code 1UEC) independently (19Yuzawa S. Suzuki N.N. Fujioka Y. Ogura K. Sumimoto H. Inagaki F. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1479-1480Crossref PubMed Scopus (11) Google Scholar, 20Yuzawa S. Suzuki N.N. Fujioka Y. Ogura K. Sumimoto H. Inagaki F. Genes Cells. 2004; 9: 443-456Crossref PubMed Scopus (53) Google Scholar). The structure of the autoinhibited form of the tandem SH3 domains reveals that elongated monomers are related by a crystallographic 2-fold axis at the hinge, forming an intertwisted dimer with a dumbbell-like shape (89 × 64 × 64 Å). The strand-exchanged region where βA, βB, and βC of the N-terminal moiety of one monomer were intertwined with βD, the 310 helical region, and βE of the other (Fig. 1, B and C) takes a typical SH3 fold. This strand-exchanged and intertwisted region was identified as the N-terminal SH3 domain, although the distal loop of the canonical SH3 fold was extended to form the hinge. The split half of the intertwisted dimer in the crystal structure has been assumed to be physiologically relevant and to represent the structure of the tandem SH3 domains in the autoinhibited form (18Groemping Y. Lapouge K. Smerdon S.J. Rittinger K. Cell. 2003; 113: 343-355Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 20Yuzawa S. Suzuki N.N. Fujioka Y. Ogura K. Sumimoto H. Inagaki F. Genes Cells. 2004; 9: 443-456Crossref PubMed Scopus (53) Google Scholar), which we called the globular module (Fig. 1B). However, a crucial issue remains to be clarified: whether the globular module exists in solution and if it represents the structure of the autoinhibited form of the tandem SH3 domains. This prompted us to investigate the structural characterization of the tandem SH3 domains in the autoinhibited form in solution. Herein, we report the structural properties of p47phox(151-340) in solution elucidated by NMR spectroscopy and small angle x-ray scattering (SAXS) analysis. Sample Preparation—The truncated form of p47phox, residues 151-340 (p47phox(151-340)) including PBR/AIR, was expressed and purified as previously reported (19Yuzawa S. Suzuki N.N. Fujioka Y. Ogura K. Sumimoto H. Inagaki F. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1479-1480Crossref PubMed Scopus (11) Google Scholar). The truncated form of p47phox, residues 151-286, corresponding to the tandem SH3 domains without PBR/AIR (p47phox(151-286)), was cloned into a pGEX-2T vector (Amersham Biosciences), transformed in Escherichia coli BL21(DE3), and expressed, as previously described (17Shiose A. Sumimoto H. J. Biol. Chem. 2000; 275: 13793-13801Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The cells were disrupted by sonication at 4 °C in 25 mm phosphate-buffered saline buffer at pH 7.4. The protein was applied to a glutathione-Sepharose 4B column (Amersham Biosciences) equilibrated with phosphate-buffered saline buffer at pH 7.4, and the bound protein was eluted using 25 mm Tris buffer, pH 8.0, and 25 mm reduced glutathione. The N-terminal glutathione S-transferase tag of p47phox(151-286) was then removed by incubation with thrombin protease (Amersham Biosciences) for 12 h at 25 °C, and the digested protein was dialyzed against 2 liters of 25 mm Tris buffer at pH 8.0. The protein was purified by anion exchange chromatography on a resource Q 6-ml column (Amersham Biosciences) equilibrated with 25 mm Tris buffer at pH 8.0, and the bound protein was eluted using a gradient of NaCl from 0 to 500 mm in the same buffer. Further purification was carried out by gel filtration chromatography on a Superdex 75 column (Amersham Biosciences) and eluted with 25 mm BisTris buffer at pH 6.5 and 150 mm NaCl. The protein was concentrated using Centriprep YM-10 (Millipore) to ∼10 mg/ml. Analytical Size Exclusion Chromatography—Size exclusion chromatography was carried out at 25 °C using a Superose 12 HR 10/30 column (Amersham Biosciences) attached to an ÄKTA system (Amersham Biosciences). Sample containing 30-120 μg of purified p47phox(151-340) was passed over the Superose 12 column equilibrated with running buffer containing 25 mm BisTris buffer at pH 6.5 and 150 mm NaCl. The samples were eluted at a flow rate of 0.5 ml/min, and the fractions were monitored by absorbance at 280 nm. The column was calibrated using the following molecular mass standards: RNase A (13.7 kDa), chicken egg white ovalbumin (43 kDa), bovine serum albumin (67 kDa), (Amersham Biosciences), and bovine carbonic anhydrase (29 kDa) (Sigma-Aldrich). A standard curve for molecular mass was constructed by plotting molecular mass against elution volume. Molecular weight for injected p47phox(151-340) was estimated in comparison with the standard curve. Analytical Ultracentrifugation—Sedimentation equilibrium experiments were carried out using a Beckman Model XL-I analytical ultracentrifuge (Beckman Coulter, Inc.) equipped with both absorbance and interference optical detection system with an An-60 Ti rotor at 25 °C. Loaded p47phox(151-340) sample concentrations were 1.5, 2.8, and 4.2 mg/ml in 25 mm BisTris buffer at pH 6.5 and 150 mm NaCl. The protein samples were 110 μl with 130 μl of reference buffer in six-channel centerpiece. The data were collected at equilibrium for three different angular velocities: 12,000, 16,000, and 20,000 rpm. An interference fringe was measured using the Rayleigh interference optics. After 12 h of centrifugation, displacement of the interference fringes was compared at 2-h intervals to ensure that the sedimentation equilibrium was reached. Data analysis was carried out using the Beckman Optima XL-A/XL-I software, version 4.1(Beckman Corlter, Inc.) based on the Origin software (Microcal, Inc.). Small Angle X-ray Scattering—SAXS data were collected on both p47phox(151-340) and p47phox(151-286) within a concentration range of the proteins from 2 to 16 mg/ml to estimate the possible effects of protein concentration on the determination of structural parameters. All of the measurements were made using a SAXS diffractometer in BL-10C installed at the Photon Factory in Tukuba, Japan (21Ueki 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, 22Kataoka M. Head J.F. Seaton B.A. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6944-6948Crossref PubMed Scopus (149) Google Scholar). The wavelength of the x-ray was 1.488 Å. The sample cell had a volume of 50 μl and a 1-mm path length with quartz windows. The data acquisition time was 600 s for each measurement. The identical buffer solution to the sample was recorded to measure solvent scattering. Protein scattering was obtained by subtracting the solvent scattering as the background trace. The scattering data were analyzed using the Guinier approximation I(Q)=I(0) exp(−Rg2Q2/3), where Q, Rg and I(0) were the momentum transfer, the radius of gyration, and the intensity at a zero scattering angle, respectively (23Guinier A. Fournet G. Small-Angle Scattering of X-ray. John Wiley & Sons, Inc., New York1955Google Scholar). Q was defined as Q = 4π sinθ/λ, where 2θ and λ were the scattering angle and wavelength of the x-rays, respectively. The I(0) and Rg values were calculated using the intensity of zero angle (ln(I(0)) and slope ((−Rg2/3)) by linear extrapolation of the Guinier plots in the range of Q × Rg < 1.8 (22Kataoka M. Head J.F. Seaton B.A. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6944-6948Crossref PubMed Scopus (149) Google Scholar, 23Guinier A. Fournet G. Small-Angle Scattering of X-ray. John Wiley & Sons, Inc., New York1955Google Scholar, 24Yuzawa S. Yokochi M. Hatanaka H. Ogura K. Kataoka M. Miura K. Mandiyan V. Schlessinger J. Inagaki F. J. Mol. Biol. 2001; 306: 527-537Crossref PubMed Scopus (51) Google Scholar). To estimate the relative molecular weight of scattering species, the zero angle intensity I(0) was scaled to the relative molecular weight using the scattering data for bovine carbonic anhydrase (Sigma-Aldrich), a monomeric protein with a molecular mass of 29 kDa. The distance distribution function, P(r), defined by P(r) = 1/2π2 ∫I(Q)Qr sin(Qr) dQ, corresponds to the distribution of distance, r, between the volume element. P(r) was calculated by the indirect Fourier transform algorithm using the GNOM program (25Svergun D.I. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2874) Google Scholar). The Q ranges used in the P(r) analysis were from 0.02 to 0.30 Å-1 for p47phox(151-340) and from 0.03 to 0.19 Å-1 for p47phox(151-286). The Rg values, calculated as Rg2=∫r2P(r)dr/(2 ∫P(r)dr), were estimated from the distance distribution function, P(r). The Dmax was a maximum dimension. Structural parameters were derived using both Guinier analysis and the distance distribution function, P(r). The distance distribution functions using the crystal structure coordinates (Protein Data Bank code 1UEC) were estimated from the frequency of distances among carbon, nitrogen, and oxygen atoms in a 1-Å interval (24Yuzawa S. Yokochi M. Hatanaka H. Ogura K. Kataoka M. Miura K. Mandiyan V. Schlessinger J. Inagaki F. J. Mol. Biol. 2001; 306: 527-537Crossref PubMed Scopus (51) Google Scholar). The Rg was calculated from the coordinates. The maximum dimension, Dmax, was derived from the point at which P(r) approaches 1/100 of the maximum intensity. Low resolution models of p47phox(151-340) were generated from experimental scattering data by the ab initio shape determination program DAMMIN (26Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1692) Google Scholar). DAMMIN calculates a volume of a protein filled with densely packed spheres (dummy atoms) to fit the experimental scattering data by a simulated annealing minimization procedure. The scattering data in the range of the momentum transfer with a Q value of 0.02-0.30 Å-1 were used for the fit. Ten independent fits were run with DAMMIN. The independent models were superimposed using the program SUPCOMB (27Kozin M.B. Svergun D.I. J. Appl. Crystallogr. 2001; 34: 33-41Crossref Scopus (1068) Google Scholar) and averaged by the program DAMAVER (28Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1578) Google Scholar), highlighting common structural features. NMR Spectroscopy—The sequence specific resonance assignments of p47phox(151-340) were previously described (29Yuzawa S. Yokochi M. Fujioka Y. Ogura K. Sumimoto H. Inagaki F. J. Biomol. NMR. 2004; (in press)PubMed Google Scholar). A NMR sample containing 1.0 mm15N-labeled p47phox (151-340) in 25 mm BisTris buffer, 150 mm NaCl, pH 6.5, in 90% H2O, 10% D2O was utilized for the steady state heteronuclear 1H-15N NOE experiments. The NOE spectra were recorded on a Varian Unity plus 600 MHz NMR spectrometer at 25 °C using a sensitivity enhanced technique with pulsed field gradients (30Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (1992) Google Scholar). Four sets of the spectra were acquired using a 3.0 s relaxation delay in the experiment. The 15N-labeled sample containing ∼0.3 mm p47phox(151-340) protein in 25 mm BisTris buffer (pH 6.5) and 150 mm NaCl was lyophilized from water and dissolved into 99.9% D2O. After leaving the sample for ∼15 min to allow temperature equilibration, 1H-15N HSQC spectra were recorded under identical conditions at different time intervals. The total measurement time for each HSQC spectrum was ∼30 min. Two NMR samples were prepared to measure 1H-15N residual dipolar couplings in 500 μl of 90% H2O/10% D2O (pH 6.5) in Wilmad sample tubes, the isotropic sample, and the aligned sample. The isotropic sample contains 1 mm protein dissolved in 25 mm BisTris and 150 mm NaCl. The aligned sample contains 0.3 mm protein dissolved in isotropic sample buffer with an additional 5% C12E5 polyethylene glycol/n-hexanol mixture with a molar ratio of surfactant to alcohol of 0.96 (31Rückert M. Otting G. J. Am. Chem. Soc. 2000; 122: 7793-7797Crossref Scopus (538) Google Scholar). The weak alignment was established throughout the measurements because the deuterium signal was observed as a sharp doublet with ∼24 Hz splitting at 25 °C before and after the measurements. One-bond 1H-15N coupling constants were measured on a Varian Unity INOVA 800 MHz NMR spectrometer at 25 °C using two-dimensional 1H-15N IPAP-type sensitivity enhanced HSQC (IPAP HSQC) spectra with in-phase (IP) or anti-phase (AP) selections (32Ottiger M. Bax A. J. Biomol. NMR. 1998; 12: 361-372Crossref PubMed Scopus (241) Google Scholar, 33Ottiger M. Delaglio F. Bax A. J. Magn. Reson. 1998; 131: 373-378Crossref PubMed Scopus (836) Google Scholar). The weighted sum and difference of the IP and AP IPAP HSQC spectra yielded spectra displaying only the up-field and down-field 15N doublet components. The residual dipolar coupling (RDC) values were obtained by subtracting the observed coupling values of the isotropic sample from those of the aligned sample. All of the two-dimensional NMR experiments were carried out using 256 and 1024 complex points in t1 and t2, respectively. Final data sets comprised 1024 and 4096 real points with a digital resolution of 2.9 and 2.7 Hz/point in F1 and F2, respectively. All of the pulse sequences were a modified version of the Varian Protein Pack (www.varianinc.com). NMR spectra were processed using VNMR (Varian Instruments, Palo Alto, CA), or NMRPipe (34Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11279) Google Scholar). TALOS software was used to predict backbone dihedral angles (35Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2727) Google Scholar). Fitting of the dipolar couplings to the structure was made using the Module program (36Dosset P. Hus J.C. Marion D. Blackledge M. J. Biomol. NMR. 2001; 20: 223-231Crossref PubMed Scopus (202) Google Scholar). All of the structure figures were prepared using PyMOL (37DeLano W.L. The PyMOL Molecular Graphics System. 2001; (www.pymol.org)Google Scholar). Molecular Mass in Solution—Because the tandem SH3 domains in the autoinhibited form of p47phox (p47phox(151-340)) exist as an intertwisted dimer in the crystalline state, we first investigated whether p47phox(151-340) could form a dimer or not in solution. Analytical size exclusion chromatography of purified p47phox(151-340) indicated that the protein forms a single species with no evidence of aggregation (Fig. 2A). Calibration with molecular weight standards revealed that p47phox(151-340) corresponded to a globular protein of ∼24 kDa, roughly consistent with the molecular mass calculated by the primary amino acid sequence (∼22 kDa). The sedimentation equilibrium experiments were subsequently performed, which give a molecular weight independent of a molecular shape. The sedimentation equilibrium data for the p47phox(151-340) at three different speeds are shown in Fig. 2B (lower panel). The data were analyzed based on the monomeric model. The goodness of fit to the model is excellent as shown in Fig. 2B (upper panel). We estimated an average molecular mass of 21400 ± 800 Da for p47phox(151-340), which is within an experimental error of the molecular mass of 21989 calculated for the p47phox(151-340) monomer from its amino acid sequence. A higher order oligomer or molecular aggregate was not detected. Molecular Mass and Average Size by Small Angle X-ray Scattering—The molecular masses and average sizes of p47phox(151-340) and p47phox(151-286) were estimated by SAXS analysis. The measured SAXS profiles showed obvious differences between p47phox(151-340) and p47phox(151-286) (Fig. 3A and 3B). Because the SAXS profile is sensitive to the size and shape of scattering molecules, the difference was attributed to the difference in the structures of the two proteins. A more quantitative representation of the structural difference can be obtained from the analyses of the Guinier approximation of the scattering data, which provides two structural parameters, the radius of gyration (Rg) and the relative molecular mass (Mr) from the zero angle scattering intensity, I(0). The small angle regions of the Guinier plots (ln(I(Q)) versus Q2) obtained from the scattering data were fitted to a single straight line (see "Experimental Procedures" for the definition of I(Q) and Q), as shown in Fig. 3 (C and D). These data sets for each protein were considered free of high molecular mass aggregates, because an upward shift in the lower angle region of the Guinier plots was not detected (22Kataoka M. Head J.F. Seaton B.A. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6944-6948Crossref PubMed Scopus (149) Google Scholar, 23Guinier A. Fournet G. Small-Angle Scattering of X-ray. John Wiley & Sons, Inc., New York1955Google Scholar, 24Yuzawa S. Yokochi M. Hatanaka H. Ogura K. Kataoka M. Miura K. Mandiyan V. Schlessinger J. Inagaki F. J. Mol. Biol. 2001; 306: 527-537Crossref PubMed Scopus (51) Google Scholar). Each of these data sets showed no concentration dependence in Rg and I(0), indicating that no concentration dependent interaction existed. The relative molecular masses of p47phox(151-340) and p47phox(151-286) were scaled using the scattering data for bovine carbonic anhydrase, a monomeric protein with a molecular mass of 29 kDa. The molecular masses of p47phox(151-340) and p47phox(151-286) were estimated to be 22.5 and 15.4 kDa, respectively. These values are in good agreement with those calculated from the amino acid sequences. Considering the molecular mass estimated from the gel filtration analysis, the sedimentation equilibrium analysis, and the small angle x-ray scattering data, we concluded that both proteins exist as monomers in solution. Interestingly, Rg of p47phox(151-340) was found to be 19.3 Å in contrast to 25.4 Å of p47phox(151-286), showing that the average molecular dimension of p47phox(151-340) is much smaller than that of p47phox(151-286) (Table I).Table ISummery of structural parameters of p47phox(151-340) and p47phox(151-286)p47phox(151-340)p47phox(151-286)Guinire approximationRg (Å)aRg is the radius of gyration, derived from the scattering data using Gunier approximation, the program GNOM, and the DAMMIN.19.3 (0.5)bThe values in parentheses indicate standard deviations of the structural parameters estimated by SAXS analysis.25.4 (0.9)Mr saxs (kDa)cMr saxs and Mr seq are molecular masses estimated from the scattering data and calculated from the primary sequence as a monomer.22.5 (0.6)15.4 (0.6)Mr seq (kDa)cMr saxs and Mr seq are molecular masses estimated from the scattering data and calculated from the primary sequence as a monomer.22.015.3P(r) analysisRg (Å)aRg is the radius of gyration, derived from the scattering data using Gunier approximation, the program GNOM, and the DAMMIN.19.2 (0.3)25.3 (0.8)Dmax (Å)dDmax is a maximum dimension.6080Ab initio shape analysiseAb inito molecular shapes were calculated with the program DAMMIN.Rg (Å)aRg is the radius of gyration, derived from the scattering data using Gunier approximation, the program GNOM, and the DAMMIN.19.32 (0.01)Dmax (Å)dDmax is a maximum dimension.59.3 (0.2)χ20.51a Rg is the radius of gyration, derived from the scattering data using Gunier approximation, the program GNOM, and the DAMMIN.b The values in parentheses indicate standard deviations of the structural parameters estimated by SAXS analysis.c Mr saxs and Mr seq are molecular masses estimated from the scattering data and calculated from the primary sequence as a monomer.d Dmax is a maximum dimension.e Ab inito molecular shapes were calculated with the program DAMMIN. Open table in a new tab Molecular Shapes and Dimensions of p47phox(151-340) and p47phox(151-286) Estimated from the Distance Distribution Function—The distance distribution function, P(r) reveals the approximate histogram for the interatomic distances between carbon, nitrogen, and oxygen atoms in a molecule and directly gives information on the shape of the molecules. Therefore, P(r) provides a quantitative evaluation of the conformational properties of proteins in solution. P(r) functions of p47phox(151-286) and p47phox(151-340) were evaluated from a set of scattering data using the indirect Fourier transform method (25Svergun D.I. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2874) Google Scholar). The observed structural parameters from the P(r) analysis are summarized in Table I together with those estimated from the Guinier approximation. The P(r) functions of p47phox(151-340) and p47phox(151-286) are shown in Fig. 3 (E and F), respectively. The P(r) of p47phox(151-340) showed a single Gaussian-like curve with a peak at 22 Å and a half-width of 28 Å. Because a Gaussian-like curve is characteristic to a spherical molecule, p47phox(151-340) was considered a globular protein (Fig. 3E). Contrastingly, the P(r) of p47phox(151-286) showed a curve with a peak located at nearly 25 Å, and the spread of the distribution curve extended to 80 Å (Fig. 3F), suggesting a relatively elongated structure. The Rg values for p47phox(1
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