| 1. |
- Meining, Winfried, et al.
(författare)
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The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6 A resolution
- 2003
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 331:5, s. 1053-1063
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Tidskriftsartikel (refereegranskat)abstract
- Riboflavin synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8(1'-D-ribityl)-lumazine, affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The N-terminal segment (residues 1-87) and the C-terminal segment (residues 98-187) form beta-barrels with similar fold and a high degree of sequence similarity. A recombinant peptide comprising amino acid residues 1-97 forms a dimer, which binds riboflavin with high affinity. Here,we report the structure of this construct in complex with riboflavin at 2.6 Angstrom resolution. It is demonstrated that the complex can serve as a model for ligand-binding in the native enzyme. The structure and riboflavin-binding mode is in excellent agreement with structural information obtained from the native enzyme from Escherichia coli and riboflavin synthase from Schizosaccharomyces pombe. The implications for the binding specificity and the regiospecificity of the catalyzed reaction are discussed.
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| 2. |
- Zhang, X F, et al.
(författare)
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A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus
- 2003
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 328:1, s. 167-182
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Tidskriftsartikel (refereegranskat)abstract
- 6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 Angstrom, 1.85 Angstrom, 2.05 Angstrom and 2.2 Angstrom, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of singlesite mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.
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| 3. |
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| 4. |
- Foloppe, N, et al.
(författare)
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Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli : Comparison with functionally related proteins
- 2001
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 310:2, s. 449-470
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Tidskriftsartikel (refereegranskat)abstract
- The chemistry of active-site cysteine residues is central to the activity of thiol-disulfide oxidoreductases of the thioredoxin superfamily. In these reactions, a nucleophilic thiolate is required, but the associated pK(a), values differ vastly in the superfamily, from less than 4 in DsbA to greater than 7 in Trx. The factors that stabilize this thiolate are, however, not clearly established. The glutaredoxins (Grxs), which are members of this superfamily, contain a Cys-Pro-Tyr-Cys motif in their active site. In reduced Grxs, the pK(a) of the N-terminal active-site nucleophilic cysteine residue is lowered significantly, and the stabilization of the corresponding thiolate is expected to influence the redox potential of these enzymes. Here, we use a combination of long molecular dynamics (MD) simulations, pK(a) calculations, and experimental investigations to derive the structure and dynamics of the reduced active site from Escherichia coli Grx3, and investigate the factors that stabilize the thiolate. Several different MD simulations converged toward a consensus conformation for the active-site cysteine residues (Cys11 and Cys14), after a number of local conformational changes. Key features of the model were tested experimentally by measurement of NMR scalar coupling constants, and determination of pK(a) values of selected residues. The pK(a) values of the Grx3 active-site residues were calculated during the MD simulations, and support the underlying structural model. The structure of Grx3, in combination with the pK(a) calculations, indicate that the pK(a) of the N-terminal active-site cysteine residue in Grx3 is intermediate between that of its counterpart in DsbA and Trx. The pK(a) values in best agreement with experiment are obtained with a low (<4) protein dielectric constant. The calculated pK(a) values fluctuate significantly in response to protein dynamics, which underscores the importance of the details of the underlying structures when calculating pK(a) values. The thiolate of Cys11 is stabilized primarily by direct hydrogen bonding with the amide protons of Tyr13 and Cys14 and the thiol proton of Cys14, rather than by long range interactions from charged groups or from a helix macrodipole. From the comparison of reduced Grx3 with other members of the thioredoxin superfamily, a unifying theme for the structural basis of thiol pK(a) differences in this superfamily begins to emerge.
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| 5. |
- Högbom, Martin, et al.
(författare)
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Crystal Structure of Conserved Domains 1 and 2 of the Human DEAD-box Helicase DDX3X in Complex with the Mononucleotide AMP
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 372:1, s. 150-159
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Tidskriftsartikel (refereegranskat)abstract
- DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central β-sheet flanked by α-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPγS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPγS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.
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| 6. |
- Jenvert, Rose-Marie, et al.
(författare)
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The flexible n-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 365:3, s. 764-772
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Tidskriftsartikel (refereegranskat)abstract
- The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in 2 stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S (Delta L11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.
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| 7. |
- Kaiser, L, et al.
(författare)
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Structural characterization of the tetrameric form of the major cat allergen Fel d 1
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 370:4, s. 714-727
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Tidskriftsartikel (refereegranskat)abstract
- Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.
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| 8. |
- Knapp, S, et al.
(författare)
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Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus
- 1996
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 264:5, s. 1132-1144
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Tidskriftsartikel (refereegranskat)abstract
- Thermal unfolding of the small hyperthermophilic DNA-binding protein Sso7d was studied by circular dichroism spectroscopy and differential scanning calorimetry. The unfolding transition can be described by a reversible two state process. Maximum stability was observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol. The heat capacity differences between the native and the heat denatured states obtained by differential scanning calorimetry (620 cal/(mol K)) and circular dichroism spectroscopy (580 cal/(mol K)) resulted in comparable values. The thermodynamic reason for the high melting temperature of Sso7d is the shallow stability curve with a broad free energy maximum, corresponding to the relatively small heat capacity change which was obtained. The calculated stability curve shows that Sso7d has, despite of its high melting temperature, an only moderate intrinsic stability, which reaches its maximum (approximate to 7 kcal/mol) at 282 K. Sso7d is particularly poorly stabilized (approximate to 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus. Sso7d has furthermore untypically low specific enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) values at convergence temperatures. No significant differences in thermal stability of the partially methylated Sso7d from Sulfolobus solfataricus and the cloned non-methylated form of the protein expressed in Escherichia coli were observed. (C) 1996 Academic Press Limited
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| 9. |
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| 10. |
- Meining, Winfried, et al.
(författare)
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The atomic structure of pentameric lumazine synthase from Saccharomyces cerevisiae at 1.85 angstrom resolution reveals the binding mode of a phosphonate intermediate analogue
- 2000
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 299:1, s. 181-197
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Tidskriftsartikel (refereegranskat)abstract
- Lumazine synthase of Saccharomyces cerevisiae is a homopentamer with a molecular weight of 90 kDa. Crystals of the recombinant enzyme with a size of up to 1.6 mm were obtained. The space group is P4(1)2(1)2 with lattice dimensions 82.9 Angstrom * 82.9 Angstrom * 300.2 Angstrom. X-ray diffraction data collected under cryogenic conditions were complete to 1.85 Angstrom resolution. The structure of the enzyme in complex with the intermediate analogue, 5-(6-D-ribitylamino-2,4-dihydroxypyrimidine-5-yl)-1-pentyl-phosphonic acid was solved via molecular replacement using the structure of the Bacillus subtilis enzyme as search model and was refined to a final X-factor of 19.8% (R-free:22.5%). The conformation of the active site ligand of the enzyme mimicks that of the Schiff base intermediate of the enzyme-catalyzed reaction. The data enable the reconstruction of the reactant topology during the early steps of the catalytic reaction. Structural determinants, which are likely to be responsible for the inability of the S. cerevisiae enzyme to form icosahedral capsids, will be discussed.
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