| 1. |
- Andersson, Charlotta S., 1979-, et al.
(författare)
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The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134:1, s. 123-125
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Tidskriftsartikel (refereegranskat)abstract
- The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.
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| 2. |
- Berntsson, Ronnie P. -A., et al.
(författare)
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Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:4, s. 2725-2735
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Tidskriftsartikel (refereegranskat)abstract
- The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.
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| 3. |
- Berntsson, Ronnie P. A., et al.
(författare)
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Structure of dual receptor binding to botulinum neurotoxin B
- 2013
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4, s. 2058-
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3-angstrom structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points.
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| 4. |
- Berntsson, Ronnie P., et al.
(författare)
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Crystal Structures of Botulinum Neurotoxin DC in Complex with Its Protein Receptors Synaptotagmin I and II
- 2013
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 21:9, s. 1602-1611
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here, we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further shows that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs.
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| 5. |
- Egeblad, Louise, et al.
(författare)
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Structural and functional studies of the human phosphoribosyltransferase domain containing protein 1
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:23, s. 4920-30
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Tidskriftsartikel (refereegranskat)abstract
- Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k(cat)/K(m)) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.
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| 6. |
- Gad, Helge, et al.
(författare)
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MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
- 2014
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221
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Tidskriftsartikel (refereegranskat)abstract
- Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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| 7. |
- Jacobson, Mark J., et al.
(författare)
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Purification, Modeling, and Analysis of Botulinum Neurotoxin Subtype A5 (BoNT/A5) from Clostridium botulinum Strain A661222
- 2011
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 77:12, s. 4217-4222
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Tidskriftsartikel (refereegranskat)abstract
- A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences-specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.
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| 8. |
- Kmiec, Beata, et al.
(författare)
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Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:40, s. E3761-E3769
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Tidskriftsartikel (refereegranskat)abstract
- Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 angstrom, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 angstrom(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.
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| 9. |
- Massad, Tariq, et al.
(författare)
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Crystal structure of the P2 C-repressor : a binder of nonpalindromic direct DNA repeats
- 2010
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:21, s. 7778-7790
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Tidskriftsartikel (refereegranskat)abstract
- As opposed to the vast majority of prokaryoticrepressors, the immunity repressor of temperateEscherichia coli phage P2 (C) recognizes nonpalindromicdirect repeats of DNA rather thaninverted repeats. We have determined the crystalstructure of P2 C at 1.8A ° . This constitutes the firststructure solved from the family of C proteins fromP2-like bacteriophages. The structure reveals thatthe P2 C protein forms a symmetric dimer orientedto bind the major groove of two consecutive turns ofthe DNA. Surprisingly, P2 C has great similarities tobinders of palindromic sequences. Nevertheless, thetwo identical DNA-binding helixes of the symmetricP2 C dimer have to bind different DNA sequences.Helix 3 is identified as the DNA-recognition motif inP2 C by alanine scanning and the importance for theindividual residues in DNA recognition is defined.A truncation mutant shows that the disorderedC-terminus is dispensable for repressor function.The short distance between the DNA-bindinghelices together with a possible interaction betweentwo P2 C dimers are proposed to be responsible forextensive bending of the DNA. The structure providesinsight into the mechanisms behind the mutants ofP2 C causing dimer disruption, temperature sensitivityand insensitivity to the P4 antirepressor.
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| 10. |
- Massad, Tariq, 1979-
(författare)
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Structural Studies of Flexible Biomolecules and a DNA-binding Protein
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each.
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