FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1991333092> ?p ?o ?g. }

• W1991333092 endingPage "41829" @default.
• W1991333092 startingPage "41822" @default.
• W1991333092 abstract "In Archaea, fibrillarin and Nop5p form the core complex of box C/D small ribonucleoprotein particles, which are responsible for site-specific 2′-hydroxyl methylation of ribosomal and transfer RNAs. Fibrillarin has a conserved methyltransferase fold and employs S-adenosyl-l-methionine (AdoMet) as the cofactor in methyl transfer reactions. Comparison between recently determined crystal structures of free fibrillarin and fibrillarin-Nop5p-AdoMet tertiary complex revealed large conformational differences at the cofactor-binding site in fibrillarin. To identify the structural elements responsible for these large conformational differences, we refined a crystal structure of Archaeoglobus fulgidus fibrillarin-Nop5p binary complex at 3.5 Å. This structure exhibited a pre-formed backbone geometry at the cofactor binding site similar to that when the cofactor is bound, suggesting that binding of Nop5p alone to fibrillarin is sufficient to stabilize the AdoMet-binding pocket. Calorimetry studies of cofactor binding to fibrillarin alone and to fibrillarin-Nop5p binary complex provided further support for this role of Nop5p. Mutagenesis and thermodynamic data showed that a cation-π bridge formed between Tyr-89 of fibrillarin and Arg-169 of Nop5p, although dispensable for in vitro methylation activity, could partially account for the enhanced binding of cofactor to fibrillarin by Nop5p. Finally, assessment of cofactor-binding thermodynamics and catalytic activities of enzyme mutants identified three additional fibrillarin residues (Thr-70, Glu-88, and Asp-133) to be important for cofactor binding and for catalysis. In Archaea, fibrillarin and Nop5p form the core complex of box C/D small ribonucleoprotein particles, which are responsible for site-specific 2′-hydroxyl methylation of ribosomal and transfer RNAs. Fibrillarin has a conserved methyltransferase fold and employs S-adenosyl-l-methionine (AdoMet) as the cofactor in methyl transfer reactions. Comparison between recently determined crystal structures of free fibrillarin and fibrillarin-Nop5p-AdoMet tertiary complex revealed large conformational differences at the cofactor-binding site in fibrillarin. To identify the structural elements responsible for these large conformational differences, we refined a crystal structure of Archaeoglobus fulgidus fibrillarin-Nop5p binary complex at 3.5 Å. This structure exhibited a pre-formed backbone geometry at the cofactor binding site similar to that when the cofactor is bound, suggesting that binding of Nop5p alone to fibrillarin is sufficient to stabilize the AdoMet-binding pocket. Calorimetry studies of cofactor binding to fibrillarin alone and to fibrillarin-Nop5p binary complex provided further support for this role of Nop5p. Mutagenesis and thermodynamic data showed that a cation-π bridge formed between Tyr-89 of fibrillarin and Arg-169 of Nop5p, although dispensable for in vitro methylation activity, could partially account for the enhanced binding of cofactor to fibrillarin by Nop5p. Finally, assessment of cofactor-binding thermodynamics and catalytic activities of enzyme mutants identified three additional fibrillarin residues (Thr-70, Glu-88, and Asp-133) to be important for cofactor binding and for catalysis. 2′-O-Methylation is one of the most frequent modifications on specific nucleotides within rRNA and other classes of RNA. In vertebrates, more than one hundred 2′-O-methyl groups have been identified in rRNA, most of which occur at highly conserved locations within functionally important regions (1Maden B.E. Prog. Nucleic Acids Res. Mol. Biol. 1990; 39: 241-303Crossref PubMed Scopus (327) Google Scholar). The methyltransferase (MTase) 1The abbreviations used are: MTase, methyltransferase; snoRNP, small ribonucleoprotein particle; MJ, Methanococcus jannaschii; AdoMet, S-adenosyl-l-methionine; AF, Archaeoglobus fulgidus; PF, Puroccocus furiosus; ITC, isothermal titration calorimetry; AdoHcy, S-adenosyl-l-homocystein.1The abbreviations used are: MTase, methyltransferase; snoRNP, small ribonucleoprotein particle; MJ, Methanococcus jannaschii; AdoMet, S-adenosyl-l-methionine; AF, Archaeoglobus fulgidus; PF, Puroccocus furiosus; ITC, isothermal titration calorimetry; AdoHcy, S-adenosyl-l-homocystein. that is responsible for 2′-O-methylation of the majority of rRNA nucleotides is the box C/D small ribonucleoprotein particle (snoRNP) which in addition to methylating rRNA also methylates mRNA (2Cavaille J. Vitali P. Basyuk E. Huttenhofer A. Bachellerie J.P. J. Biol. Chem. 2001; 276: 26374-26383Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 3Dennis P.P. Omer A. Lowe T. Mol. Microbiol. 2001; 40: 509-519Crossref PubMed Scopus (112) Google Scholar) and small nuclear RNA (4Tollervey D. Kiss T. Curr. Opin. Cell Biol. 1997; 9: 337-342Crossref PubMed Scopus (375) Google Scholar, 5Terns M.P. Terns R.M. Gene Expr. 2002; 10: 17-39PubMed Google Scholar, 6Maxwell E.S. Fournier M.J. Annu. Rev. Biochem. 1995; 64: 897-934Crossref PubMed Scopus (536) Google Scholar, 7Weinstein L.B. Steitz J.A. Curr. Opin. Cell Biol. 1999; 11: 378-384Crossref PubMed Scopus (249) Google Scholar) and processes precursor rRNA into mature rRNAs.Box C/D snoRNPs require assembly of box C/D snoRNAs with a set of nucleolar proteins that include fibrillarin (Nop1p in yeast), Nop56/58p, and 15.5-kDa proteins (Snu13p in yeast). In Archaea, homologs of box C/D snoRNPs (box C/D sRNPs) consist of box C/D sRNAs, fibrillarin, Nop5p (a single homolog of Nop56/58p), and L7Ae proteins (3Dennis P.P. Omer A. Lowe T. Mol. Microbiol. 2001; 40: 509-519Crossref PubMed Scopus (112) Google Scholar, 8Omer A.D. Ziesche S. Decatur W.A. Fournier M.J. Dennis P.P. Mol. Microbiol. 2003; 48: 617-629Crossref PubMed Scopus (79) Google Scholar). The box C/D RNAs are responsible for the recognition of methylation target RNAs via base pairing of their antisense regions with the target. The s(no)RNP proteins contain the actual methyltransferase activity. The protein subunit that catalyzes the methyl transfer reactions in s(no)RNPs is believed to be fibrillarin. The first clue that linked the methylation activity of s(no)RNPs to fibrillarin came from the work of Tollervey et al. (9Tollervey D. Lehtonen H. Jansen R. Kern H. Hurt E.C. Cell. 1993; 72: 443-457Abstract Full Text PDF PubMed Scopus (406) Google Scholar) on yeast, where the nop1.3 allele, a temperature-sensitive mutant of the nop1 gene coding for yeast fibrillarin, exhibited a strongly inhibited precursor rRNA methylation phenotype at non-permissive temperatures. The second evidence came from the crystal structure of an archaeal fibrillarin homolog from Methanococcus jannaschii (MJ), which revealed the conservation of the AdoMet-binding fold that is common to all known MTase structures (10Wang H. Boisvert D. Kim K.K. Kim R. Kim S.H. EMBO J. 2000; 19: 317-323Crossref PubMed Scopus (143) Google Scholar). We recently determined a co-crystal structure of fibrillarin complexed with Nop5p from Archaeoglobus fulgidus (AF), which contained a bound AdoMet at the predicted binding site in fibrillarin (11Aittaleb M. Rashid R. Chen Q. Palmer J.R. Daniels C.J. Li H. Nat. Struct. Biol. 2003; 10: 256-263Crossref PubMed Scopus (106) Google Scholar). This result establishes further the role of fibrillarin as the methyltransferase.Similar to the previously known AdoMet-dependent MTases, fibrillarin contains the same set of conserved motifs that are important to the binding of AdoMet (see Fig. 1). These include motifs I-IV at the carboxyl ends of beta strands β1–β4 as defined by Cheng and Roberts (12Cheng X. Roberts R.J. Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (395) Google Scholar) in studying DNA MTases. Consistent with their important functional roles in catalysis, three temperature-sensitive mutants of yeast fibrillarin occurred within the conserved AdoMet-binding motifs. For instance, nop1.2, nop1.3, and nop1.7 mutations occurred in motif III, motif I, and motifs II and IV, respectively (9Tollervey D. Lehtonen H. Jansen R. Kern H. Hurt E.C. Cell. 1993; 72: 443-457Abstract Full Text PDF PubMed Scopus (406) Google Scholar). Furthermore, by using an in vitro reconstituted archaeal sRNP enzyme, Omer et al. (13Omer A.D. Ziesche S. Ebhardt H. Dennis P.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5289-5294Crossref PubMed Scopus (158) Google Scholar) demonstrated that substitutions of two amino acids within motifs I and III of Sulfolobus solfataricus fibrillarin (A85V and P129V) resulted in completely or partially abolished methylation activities, whereas sRNP assembly was unaffected. These data suggest that fibrillarin has a function similar to other previously known AdoMet-dependent MTases.However, recent structural and biochemical evidence suggests that fibrillarin alone binds weakly to AdoMet. There are currently three known fibrillarin crystal structures, all from Archaea, MJ (10Wang H. Boisvert D. Kim K.K. Kim R. Kim S.H. EMBO J. 2000; 19: 317-323Crossref PubMed Scopus (143) Google Scholar), AF (11Aittaleb M. Rashid R. Chen Q. Palmer J.R. Daniels C.J. Li H. Nat. Struct. Biol. 2003; 10: 256-263Crossref PubMed Scopus (106) Google Scholar), and Puroccocus furiosus (PF) (14Deng L. Starostina N.G. Liu Z.J. Rose J.P. Terns R.M. Terns M.P. Wang B.C. Biochem. Biophys. Res. Commun. 2004; 315: 726-732Crossref PubMed Scopus (17) Google Scholar). Among these three structures, only that in complex with AF Nop5p had the cofactor bound at the predicted AdoMet-binding site. Interestingly, in both structures of fibrillarin without the bound Nop5p (MJ and PF), motif I and the loop connecting β1 and α1 of fibrillarin adopted conformations that would exclude the binding of AdoMet (10Wang H. Boisvert D. Kim K.K. Kim R. Kim S.H. EMBO J. 2000; 19: 317-323Crossref PubMed Scopus (143) Google Scholar, 14Deng L. Starostina N.G. Liu Z.J. Rose J.P. Terns R.M. Terns M.P. Wang B.C. Biochem. Biophys. Res. Commun. 2004; 315: 726-732Crossref PubMed Scopus (17) Google Scholar). This clearly raised the possibility that Nop5p has a functional role in cofactor binding by modulating the conformation of AdoMet-binding motifs in fibrillarin. However it is also possible that the binding of the cofactor itself induces the conformational change of the cofactor binding pocket in fibrillarin.In the co-crystal structure of AF fibrillarin-Nop5p complex bound with AdoMet (holocomplex), a number of conserved fibrillarin residues were observed to interact directly with AdoMet (Fig. 1). Glu-88 forms two hydrogen bonds with the ribose hydroxyl groups of AdoMet. Thr-70 also forms a hydrogen bond with the carboxyl group of AdoMet. Asp-133 is situated near the positively charged thiomethyl group and thus may facilitate cofactor binding through favorable electrostatic interactions. Finally, Tyr-89 establishes an aromatic stacking interaction with the adenine ring of the cofactor. The opposing side of the phenol ring of Tyr-89 closely packs against a positive Nop5p residue, Arg-169. This structural arrangement between Tyr-89 of fibrillarin and Arg-169 of Nop5p creates a strong cation-π interaction as evident from the large negative electrostatic and van der Waals energies between them (Ees = –5.2 kcal/mol, Evdw =–2.8 kcal/mol) (computed by CAPTURE) (15Gallivan J.P. Dougherty D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9459-9464Crossref PubMed Scopus (1670) Google Scholar). Notably, Tyr-89 would be completely exposed to solvent without forming the cation-π interaction. Tyr-89 is well conserved among all known fibrillarin proteins. Arg-169 is also conserved among the Nop family of proteins, suggesting that this cation-π interaction is preserved in all homologous complexes of fibrillarin and Nop5p. The importance of this cation-π bridge formed between fibrillarin and Nop5p may play a critical role in stabilizing the association of the cofactor with fibrillarin.In this work, we examined the requirement of Nop5p for cofactor binding by combining structural, thermodynamic, and functional analyses. To discern the effect on fibrillarin conformational changes in the cofactor binding pocket, we refined the crystal structure of Nop5p-fibrillarin complex without soaked cofactors at 3.5 Å. We also established isothermal titration calorimetry conditions, which allowed us to directly compare thermodynamic parameters of AdoMet binding to different fibrillarin complexes. By monitoring the changes in heat as the cofactor was titrated into a protein solution, we obtained binding constants and enthalpy changes of AdoMet binding to the Nop5p-fibrillarin complex, to mutant fibrillarin Nop5p complexes, and to fibrillarin. Furthermore, to assess the functional importance of the cofactor interacting residues in both fibrillarin and Nop5p, we compared in vitro methylation activities of the wild-type Nop5p-fibrillarin complex and its mutants where several AdoMet-binding residues were disrupted. These structural, thermodynamic, and functional studies support the stabilizing role of Nop5p in cofactor binding to fibrillarin and clearly identify residues in fibrillarin that are directly involved in cofactor binding and catalysis.EXPERIMENTAL PROCEDURESProtein Purification—The wild-type Nop5p-fibrillarin protein complex and fibrillarin alone were purified as described previously by Aittaleb et al. (11Aittaleb M. Rashid R. Chen Q. Palmer J.R. Daniels C.J. Li H. Nat. Struct. Biol. 2003; 10: 256-263Crossref PubMed Scopus (106) Google Scholar). All the mutations were performed within co-expressing fibrillarin and Nop5p genes using the QuikChange mutagenesis kit from Stratagene. The mutants were purified in a similar procedure as that for the wild type. All proteins were subjected to gel filtration on a Superdex S200 column (Amersham Biosciences) in a buffer containing 20 mm Tris, pH 8.0, 5% glycerol, 1.0 m NaCl, 5 mm β-mercaptoethanol, and 0.5 mm EDTA. Gel filtration profiles for all mutant fibrillairin-Nop5p complexes were similar to that of the wild-type complex, suggesting no misfolding of the mutant proteins.Structure Refinement—During earlier structure determination of the fibrillarin-Nop5p complex, we collected a multiple wavelength anomalous diffraction data set from a crystal of fibrillarin-Nop5p complex containing seleno-methionine without soaked cofactors. This data set allowed phase determination. The final structure, however, was refined against the diffraction data set collected from cofactor-soaked fibrillarin-Nop5p crystal because of its higher resolution. To elucidate the structural conformation around the cofactor binding site in the absence of a bound cofactor, we have now refined the coordinates of fibrillarin-Nop5p complex against the data set collected at the selenine K-edge. This data set has the best statistics among the three data sets (11Aittaleb M. Rashid R. Chen Q. Palmer J.R. Daniels C.J. Li H. Nat. Struct. Biol. 2003; 10: 256-263Crossref PubMed Scopus (106) Google Scholar). Refinement was carried out using crystallography NMR software (CNS) (16Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar) by including all 10 selenine atoms and keeping the Bijvoet pairs separated. The statistics of structural refinement are listed in Table I. Structure comparison of the apocomplex with earlier structures is presented in Fig. 2.Table IRefinement statistics of the apocomplex of Nop5p-fibrillarinResolution range (Å)500.0-3.5R factor (%)29.8Rfree (%)32.3Model informationNumber of monomers1Number of amino acid residues445Number of protein atoms3642Number of waters0Average atomic B factors (Å2)Main chain71.6Side chain74.5Root mean square deviation of the modelBond length (Å)0.006Bond angle (°)1.2B, bonded main chain (Å2)1.4B, bonded side chain (Å2)1.8Ramachandran plotMost favored (%)316Allowed (%)83Generously allowed (%)4Disallowed (%)1 Open table in a new tab Fig. 2Structural comparison of fibrillarin suggests large conformational changes at the cofactor binding site.A, the holocomplex of fibrillarin-Nop5p (AF fibrillarin (blue) and AF Nop5p (green)) was superimposed with the apocomplex (AF fibrillarin (orange) and AF Nop5p (gray)). SigmaA-weighted 3Fo – 2Fc map at 1.0 σ was displayed at the cofactor binding site for the apocomplex, indicating the absence of a bound cofactor molecule. B, the holocomplex of fibrillarin-Nop5p (AF fibrillarin (blue) and AF Nop5p (green)) was superimposed with PH fibrillarin (gray). Phe-106 of PH fibrillarin is highlighted and labeled. C, the holocomplex of fibrillarin-Nop5p (AF fibrillarin (blue) and AF Nop5p (green)) was superimposed with MJ fibrillarin (cyan). Tyr-106 of MJ fibrillarin is highlighted and labeled.View Large Image Figure ViewerDownload (PPT)Isothermal Titration Calorimetry—Titration experiments were performed by isothermal titration calorimetry (ITC) using a VP-ITC microcalorimeter (Microcal, Inc., Northampton, MA) interfaced with a computer. The titration calorimeter consists of 1.45 ml of sample cell containing a macromolecule solution and a matched thermal reference cell filled with water. AdoMet or S-adenosyl-l-homocystein (AdoHcy) was dissolved in the same buffer used for the protein to be titrated. Prior to the experiment, samples were filtered and degassed under vacuum for 10 min in a Thermo Vac system (Microcal). The sample cell was filled with the working buffer (for dilution heat control) or with the protein to be characterized. Titrations with the cofactor (25× protein concentration) consisted of a preliminary 2-μl injection (not to be considered in data analysis) followed by twenty-nine 5 μl injections with at least 4-min intervals between injections. All runs were made at constant stirrer speed of 310 rpm, and all experiments were performed at 30 °C. The heat caused by the dilution of the cofactor was subtracted from the experimental data conducted with the protein. Protein concentrations were determined by UV absorption using the theoretical extinction coefficients computed from the amino acid sequences (ϵ280 = 48360 m–1 cm–1). The evolved heat peaks were integrated and then fitted to a theoretical titration curve of a single binding site model by non-linear least squares to yield ΔH0 (molar enthalpy change in kcal/mol), Ka (binding constant in molar–1) and n (stoichiometry ratio).All isotherms show negative deflection indicative of an exothermic reaction (see Fig. 3, A and B). A separate run with cofactor titrating into buffer showed insignificant endothermic and equal sized dilution peaks, suggesting that at this concentration no aggregation of the cofactor in buffer occurs (Fig. 3B). Despite the fact that Nop5p-fibrillarin complex forms a homodimer as previously observed in crystal and in solution (11Aittaleb M. Rashid R. Chen Q. Palmer J.R. Daniels C.J. Li H. Nat. Struct. Biol. 2003; 10: 256-263Crossref PubMed Scopus (106) Google Scholar, 18Rashid R. Aittaleb M. Chen Q. Spiegel K. Demeler B. Li H. J. Mol. Biol. 2003; 333: 295-306Crossref PubMed Scopus (58) Google Scholar), ITC data clearly exhibited the characteristics of single set of identical sites. Therefore, all data were processed using the standard One Set of One Site model as implemented in the Microcal Origin software based on the Wiseman isotherm (17Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2416) Google Scholar),Q=nMtΔH0V02[1+XtnMt+1nKaMt−(1+XtnMt+1nKaMt)2−4XtnMt]eq.1 where Q is the heat content of the solution, Ka is the binding constant, and Mt is the bulk concentration of the protein in volume V0, Xt is the bulk concentration of the ligand, and n is the number of binding sites. During actual fitting, the heat released for each injection increment was used with the correction of displaced volume caused by each injection. Therefore, the enthalpy change (ΔH0) and the binding constant (Ka) were directly obtainable from the experiments after data processing. The free energy and entropy were subsequently calculated using ΔG0 = –RTlnKa and TΔS0 = –ΔG0+ΔH0, respectively. The resulting binding parameters (binding constant Ka, molar Gibbs free energy, ΔG0, molar enthalpy ΔH0, and molar entropy ΔS0) of the proteins with AdoMet and with AdoHcy (D133A mutant only), and the standard deviations are summarized in Table II.Fig. 3Example ITC profiles. In both A and B profiles, the upper panels indicate the baseline-subtracted raw titration data obtained for injections of the cofactor into the protein solution, and the lower panels indicate the integrated areas for the peaks normalized to the cofactor/protein molar ratio. Dark dots showing the experimental data and solid lines showing the best fit to one set of the binding sites model were obtained by nonlinear least squares fitting. A, titration of AF fibrillarin with AdoMet. B, titration of the D133A mutant complex of Nop5p-fibrillarin with AdoHcy. Top titration also includes injections of AdoMet into buffer.View Large Image Figure ViewerDownload (PPT)Table IIThermodynamic parameters for AdoMet binding to fibrillarin and Nop5p-fibrillarin complexesKaΔH0KdΔG0TΔS0105 (m-1)kcal/molμmkcal/molkcal/molFibrillarin0.95 ± 0.09-9.1 ± 0.210.5 ± 1-6.87 ± 0.06-2.23 ± 0.1Wild type Nop5p-fibrillarin3.7 ± 0.3-11.7 ± 0.32.7 ± 0.2-7.69 ± 0.05-4.01 ± 0.3Y89A0.55 ± 0.09-4.65 ± 0.218.1 ± 1-6.55 ± 0.061.9 ± 0.1E88A6.8 ± 0.8-13.2 ± 0.51.4 ± 0.2-8.05 ± 0.07-5.15 ± 0.4T70A6.2 ± 0.4-9.7 ± 0.11.6 ± 0.1-8.00 ± 0.04-1.7 ± 0.06D133A0.11 ± 0.005-5.4 ± 0.290.0 ± 4-5.58 ± 0.030.18 ± 0.2D133A (AdoHcy)0.67 ± 0.04-8.84 ± 0.00314.9 ± 0.9-6.66 ± 0.03-2.17 ± 0.03R169A (Nop5p)3.7 ± 0.2-8.5 ± 0.12.7 ± 0.1-7.69 ± 0.03-0.81 ± 0.07 Open table in a new tab Electrostatic Potential Calculations—To assess the electrostatic contribution of Asp133 to cofactors binding, electrostatic potentials were calculated with the hybrid boundary element and finite difference nonlinear Poisson-Boltzmann (PB) algorithm (19Boschitsch A.H. Fenley M.O. J. Comput. Chem. 2004; 25: 935-955Crossref PubMed Scopus (83) Google Scholar). The atomic coordinates of the wild-type Nop5p-fibrillarin complex (Protein Data Bank accession number 1NT2) were employed, and no missing residues or hydrogen atoms were added. For the D133A mutant Nop5p-fibrillarin complex, the Asp-133 residue was substituted for alanine followed by stereochemistry regularization using the O program (20Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). The dielectric constant of the solute was set to 2 and the solvent to 80. The temperature and ionic strength of the solution was fixed at 298 Kelvin and 0.1 m NaCl, respectively. The Parse parameter set (21Sitkoff D. Sharp K.A. Honig B. J. Phys. Chem. 1994; 98: 1978-1988Crossref Scopus (1838) Google Scholar) was used to assign van der Waals radii to atoms. The formal charge set was employed with a charge of –1e assigned to aspartate, glutamate, and the C-terminal residues. A charge of +1e was assigned to lysine and arginine residues. Histidine residues were assigned a charge of +0.5e. The net charge of the wild-type and mutant Nop5p-fibrillarin complexes is +6.5e and +7.5e, respectively. The solvent-excluded molecular surface was employed to define the solute-solvent boundary based on a 1.4-Å solvent probe radius. No ion exclusion region was considered.The MSMS molecular surface program (22Sanner M.F. Olson A.J. Spehner J.C. Biopolymers. 1996; 38: 305-320Crossref PubMed Google Scholar) was employed to triangulate the solvent-excluded surface and thus generate the boundary elements (∼90,000 triangles) used in the linear PB solution. The finite difference cubic grid contained 1283 nodes. The total extent of the three-dimensional uniform Cartesian grid is four times the largest dimension of the solute. Computed electrostatic potential surfaces for both the wild-type and the D133A mutant are displayed in Fig. 4.Fig. 4Computed surface electrostatic potentials of the wild-type (left) and the D133A mutant (right) Nop5p-fibrillarin complexes at the AdoMet-binding pocket suggest electrostatic stabilization of cofactor binding by Asp133. The bound cofactor, AdoMet, is shown in stick models. The electrostatic potential was obtained by solving the nonlinear Poisson-Boltzmann equation. The solvent-excluded surface with a solvent probe radius of 1.4 Å was used to define the solute-solvent boundary. The electrostatic potential ranges from yellow to red to white to blue to green, where yellow and red are negative potential, white is neutral, and blue and green are positive potential. The electrostatic potential range is –5 kcal/mol/e (yellow) to +5 kcal/mol/e (green).View Large Image Figure ViewerDownload (PPT)In Vitro Methylation Assay—In vitro methylation assays were carried out according to a published procedure described previously (18Rashid R. Aittaleb M. Chen Q. Spiegel K. Demeler B. Li H. J. Mol. Biol. 2003; 333: 295-306Crossref PubMed Scopus (58) Google Scholar). Briefly, all enzymes were prepared to be free of ribonuclease contamination before being assembled for catalysis. Tritium-AdoMet (Sigma) was used as the methyl donor in the reaction, which permitted us to monitor the methylation reaction by counting the retained radioactivity caused by the target RNA on DE81 ion exchange filter. Wild-type Nop5p-fibrillarin or a mutant was mixed with L7Ae protein prior to being added to a pre-annealed RNA mixture containing both the AF sR3 box C/D guide RNA (rna.wustl.edu/snoRNAdb/) and a complementary target RNA oligo to the guide sequence upstream of box D sequences. The radioactivity retained on the DE81 filter at each reaction time point was quantified by scintillation counter. The production of methylation product was plotted in Fig. 5 along with least-square fitted progress curves.Fig. 5Methylation progression curves obtained from in vitro methylation assay on wild-type and mutant Nop5p-fibrillarin complexes. Each reaction mixtures contained Nop5p-fibrillarin or a mutant complex, L7Ae protein, box C/D guide RNA, target RNA oligo complementary to box C/D, and H3-labeled AdoMet cofactor. The incorporation of the methyl group was monitored by counting the retained radioactivity on extensively washed DE81 ion exchange filters at different intervals of time. The experimentally measured activities were normalized to be zero at t = 0 min and were fitted to a typical progress curve.View Large Image Figure ViewerDownload (PPT)RESULTSStructural Comparison of AdoMet-binding Sites in Nop5p-free and Nop5p-bound Fibrillarins—The fibrillarin portion of the AF Nop5p-fibrillarin complex structure (11Aittaleb M. Rashid R. Chen Q. Palmer J.R. Daniels C.J. Li H. Nat. Struct. Biol. 2003; 10: 256-263Crossref PubMed Scopus (106) Google Scholar) was superimposed with those of MJ fibrillarin (10Wang H. Boisvert D. Kim K.K. Kim R. Kim S.H. EMBO J. 2000; 19: 317-323Crossref PubMed Scopus (143) Google Scholar) and of PF fibrillarin (14Deng L. Starostina N.G. Liu Z.J. Rose J.P. Terns R.M. Terns M.P. Wang B.C. Biochem. Biophys. Res. Commun. 2004; 315: 726-732Crossref PubMed Scopus (17) Google Scholar). The backbone root mean square differences were 1.095 Å (191 Cα atoms) for MJ fibrillarin and 0.934 Å (144 Cα atoms) for PH fibrillarin. The most significant difference within the core structure of the three fibrillarins is in motif I and II where the cofactor binds (Fig. 2, B and C). In particular, the backbone geometry of motif II loop in the two free fibrillarin structures differ from each other and from the fibrillarin bound with Nop5p and AdoMet. Each free fibrillarin adopted a conformation that would exclude the binding of the cofactor. In both structures, the loops connecting β2 and α2 traversed through where the ribose moiety of AdoMet would lie. Most dramatically, the aromatic residues (Phe-106 in PF fibrillarin and Tyr-106 in MJ fibrillarin) completely swung away from where they could establish the favorable base stacking interactions with the cofactor adenine ring and protruded into the solvent region (Fig. 2, B and C). This striking difference in backbone geometry and key binding residues between the two free fibrillarin structures and that bound with cofactor suggested an intrinsic structural flexibility of this region in fibrillarin that could potentially hinder the optimal binding of the cofactor.The Apocomplex Structure of Nop5p-Fibrillarin Closely Resembles the Holocomplex—To discern whether the observed conformational differences between free fibrillarin structures and that bound with cofactor and Nop5p are induced by Nop5p binding or by cofactor binding, we refined the crystal structure of Nop5p-fibrillarin in the absence of soaked AdoMet (apocomplex). The refined apocomplex of Nop5p-fibrillarin was superimposed with the holocomplex reported previously (11Aittaleb M. Rashid R. Chen Q. Palmer J.R. Daniels C.J. Li H. Nat. Struct. Biol. 2003; 10: 256-263Crossref PubMed Scopus (106) Google Scholar). SigmaA-weighted 3Fo – 2Fc and Fo – Fc maps were computed by using the observed amplitude and phases resulted from refined coordinates. Both maps clearly showed an absence of a bound cofactor at the predicted AdoMet binding site (Fig. 2A). Fig. 2A also showed that the fibrillarin residues covering the AdoMet binding site overl" @default.
• W1991333092 created "2016-06-24" @default.
• W1991333092 creator A5005951988 @default.
• W1991333092 creator A5006302733 @default.
• W1991333092 creator A5047629405 @default.
• W1991333092 creator A5064204211 @default.
• W1991333092 date "2004-10-01" @default.
• W1991333092 modified "2023-09-27" @default.
• W1991333092 title "Structural and Thermodynamic Evidence for a Stabilizing Role of Nop5p in S-Adenosyl-L-methionine Binding to Fibrillarin" @default.
• W1991333092 cites W1556119657 @default.
• W1991333092 cites W1894304989 @default.
• W1991333092 cites W1965401154 @default.
• W1991333092 cites W1967979896 @default.
• W1991333092 cites W1970913370 @default.
• W1991333092 cites W1994711663 @default.
• W1991333092 cites W1995017064 @default.
• W1991333092 cites W2009193892 @default.
• W1991333092 cites W2013083986 @default.
• W1991333092 cites W2018512856 @default.
• W1991333092 cites W2020357919 @default.
• W1991333092 cites W2030406803 @default.
• W1991333092 cites W2031765531 @default.
• W1991333092 cites W2034407986 @default.
• W1991333092 cites W2039945618 @default.
• W1991333092 cites W2054525948 @default.
• W1991333092 cites W20619123 @default.
• W1991333092 cites W2063098789 @default.
• W1991333092 cites W2066162276 @default.
• W1991333092 cites W2075369230 @default.
• W1991333092 cites W2080823722 @default.
• W1991333092 cites W2090951806 @default.
• W1991333092 cites W2097343134 @default.
• W1991333092 cites W2140184534 @default.
• W1991333092 cites W4293003482 @default.
• W1991333092 doi "https://doi.org/10.1074/jbc.m406209200" @default.
• W1991333092 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15286083" @default.
• W1991333092 hasPublicationYear "2004" @default.
• W1991333092 type Work @default.
• W1991333092 sameAs 1991333092 @default.
• W1991333092 citedByCount "29" @default.
• W1991333092 countsByYear W19913330922012 @default.
• W1991333092 countsByYear W19913330922013 @default.
• W1991333092 countsByYear W19913330922014 @default.
• W1991333092 countsByYear W19913330922016 @default.
• W1991333092 countsByYear W19913330922017 @default.
• W1991333092 countsByYear W19913330922018 @default.
• W1991333092 countsByYear W19913330922019 @default.
• W1991333092 countsByYear W19913330922021 @default.
• W1991333092 countsByYear W19913330922022 @default.
• W1991333092 crossrefType "journal-article" @default.
• W1991333092 hasAuthorship W1991333092A5005951988 @default.
• W1991333092 hasAuthorship W1991333092A5006302733 @default.
• W1991333092 hasAuthorship W1991333092A5047629405 @default.
• W1991333092 hasAuthorship W1991333092A5064204211 @default.
• W1991333092 hasBestOaLocation W19913330921 @default.
• W1991333092 hasConcept C104317684 @default.
• W1991333092 hasConcept C12554922 @default.
• W1991333092 hasConcept C185592680 @default.
• W1991333092 hasConcept C2776007122 @default.
• W1991333092 hasConcept C2780912031 @default.
• W1991333092 hasConcept C515207424 @default.
• W1991333092 hasConcept C55493867 @default.
• W1991333092 hasConcept C67705224 @default.
• W1991333092 hasConcept C71240020 @default.
• W1991333092 hasConcept C86803240 @default.
• W1991333092 hasConcept C95444343 @default.
• W1991333092 hasConceptScore W1991333092C104317684 @default.
• W1991333092 hasConceptScore W1991333092C12554922 @default.
• W1991333092 hasConceptScore W1991333092C185592680 @default.
• W1991333092 hasConceptScore W1991333092C2776007122 @default.
• W1991333092 hasConceptScore W1991333092C2780912031 @default.
• W1991333092 hasConceptScore W1991333092C515207424 @default.
• W1991333092 hasConceptScore W1991333092C55493867 @default.
• W1991333092 hasConceptScore W1991333092C67705224 @default.
• W1991333092 hasConceptScore W1991333092C71240020 @default.
• W1991333092 hasConceptScore W1991333092C86803240 @default.
• W1991333092 hasConceptScore W1991333092C95444343 @default.
• W1991333092 hasIssue "40" @default.
• W1991333092 hasLocation W19913330921 @default.
• W1991333092 hasOpenAccess W1991333092 @default.
• W1991333092 hasPrimaryLocation W19913330921 @default.
• W1991333092 hasRelatedWork W1499380138 @default.
• W1991333092 hasRelatedWork W1534701699 @default.
• W1991333092 hasRelatedWork W1554285585 @default.
• W1991333092 hasRelatedWork W1560687854 @default.
• W1991333092 hasRelatedWork W1965996509 @default.
• W1991333092 hasRelatedWork W1970974546 @default.
• W1991333092 hasRelatedWork W1996140555 @default.
• W1991333092 hasRelatedWork W2086466873 @default.
• W1991333092 hasRelatedWork W2127372326 @default.
• W1991333092 hasRelatedWork W2131313694 @default.
• W1991333092 hasVolume "279" @default.
• W1991333092 isParatext "false" @default.
• W1991333092 isRetracted "false" @default.
• W1991333092 magId "1991333092" @default.
• W1991333092 workType "article" @default.