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  • 1. Antuch, W
    et al.
    Berndt, Kurt D
    Eidgenössische Technische Hochschule–Hönggerberg, Zürich, Switzerland.
    Chávez, M A
    Delfín, J
    Wüthrich, K
    The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus.1993In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 212, 675-684 p.Article in journal (Refereed)
    Abstract [en]

    The solution structure of a 55-amino-acid Kunitz-type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence-specific 1H-NMR assignments were obtained at pH 4.6 and 36 degrees C, and stereo-specific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root-mean-square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, C alpha and C', and 106 pm for all heavy atoms of residues 2-53. This high-quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energy-minimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three-dimensional structure is also available.

  • 2. Benach, J
    et al.
    Filling, C
    Oppermann, U C T
    Roversi, P
    Bricogne, G
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Jörnvall, H
    Ladenstein, R
    Structure of bacterial 3 beta/17 beta-hydroxysteroid dehydrogenase at 1.2 angstrom resolution: A model for multiple steroid recognition2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 50, 14659-14668 p.Article in journal (Refereed)
    Abstract [en]

    The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Conramonas testosteroni. It catalyzes the reversible reduction/ dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids. Crystallographic analysis at 1.2 Angstrom resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry. This is one of the largest oligomeric structures refined at this resolution. The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices. The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR). Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism. In contrast to other SDR apostructures determined, the substrate binding loop is well-defined. Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.

  • 3.
    Berndt, Kurt D
    University of Chicago, USA.
    Design, synthesis, and characterization of amphiphilic helical peptides as models of protein structure1989Doctoral thesis, monograph (Other academic)
  • 4.
    Berndt, Kurt D
    et al.
    Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Beunink, J
    Schröder, W
    Wüthrich, K
    Designed replacement of an internal hydration water molecule in BPTI: structural and functional implications of a glycine-to-serine mutation.1993In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 32, 4564-4570 p.Article in journal (Refereed)
    Abstract [en]

    The three-dimensional structure of the basic pancreatic trypsin inhibitor (BPTI) contains four internal water molecules, which form a total of nine intermolecular hydrogen bonds with the BPTI polypeptide chain. To investigate the effect of such internal hydration on protein structure and stability, we displaced one of the internal water molecules in a recombinant BPTI analogue, BPTI(G36S), in which Gly 36 is replaced by serine. The replacement of a water molecule by the seryl side chain was established by the absence of the protein-water nuclear Overhauser effects (NOE) that had been attributed to the water molecule near Gly 36 in wild-type BPTI and by the presence of new, intramolecular NOEs to the hydroxyl proton of Ser 36. BPTI(G36S) has slightly reduced thermal stability compared to BPTI, corresponding to a destabilization by delta (delta G) approximately 0.7 kcal/M in 6 M guanidinium hydrochloride solution. Additionally, the stabilities of the complexes formed between BPTI(G36S) and trypsin, plasmin, or kallikrein are significantly reduced when compared to the corresponding complexes with wild-type BPTI.

  • 5.
    Berndt, Kurt D
    et al.
    Eidgenossische TH-Honggerberg, Zürich, Switzerland.
    Güntert, P
    Wüthrich, K
    Nuclear-Magnetic-Resonance Solution Structure of Dendrotoxin-K from the Venom of Dendroaspis-Polylepis-Polylepis1993In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 234, no 3, 735-750 p.Article in journal (Refereed)
  • 6.
    Berndt, Kurt D
    et al.
    Eidgenösische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Güntert, Peter
    Orbons, Leonard P.M.
    Wüthrich, Kurt
    Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures1992In: Journal of Molecular Biology, Vol. 227, 757-775 p.Article in journal (Refereed)
    Abstract [en]

    A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.

  • 7.
    Berndt, Kurt D
    et al.
    Eidgenössische TH-Hönggerberg, Zürich, Switzerland.
    Güntert, Peter
    Wüthrich, Kurt
    Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water1996In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 24, 304-313 p.Article in journal (Refereed)
    Abstract [en]

    The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using "realistic" potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.

  • 8. Brunne, R M
    et al.
    Berndt, Kurt D
    ETH Hönggerberg, Zürich, Switzerland.
    Güntert, P
    Wüthrich, K
    van Gunsteren, W F
    Güntert, P
    Wüthrich, K
    Van Gunsteren, W F
    Structure and internal dynamics of the bovine pancreatic trypsin inhibitor in aqueous solution from long-time molecular dynamics simulations1995In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 23, no 1, 49-62 p.Article in journal (Refereed)
    Abstract [en]

    Structural and dynamic properties of bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution are investigated using two molecular dynamics (MD) simulations: one of 1.4 ns length and one of 0.8 ns length in which atom-atom distance bounds derived from NMR spectroscopy are included in the potential energy function to make the trajectory satisfy these experimental data more closely. The simulated properties of BPTI are compared with crystal and solution structures of BPTI, and found to be in agreement with the available experimental data. The best agreement with experiment was obtained when atom-atom distance restraints were applied in a time-averaged manner in the simulation. The polypeptide segments found to be most flexible in the MD simulations coincide closely with those showing differences between the crystal and solution structures of BPTI.

  • 9. Caballero-Herrera, A
    et al.
    Nordstrand, Kerstin
    Södertörn University, School of Life Sciences.
    Berndt, Kurt D
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Nilsson, L
    Effect of urea on peptide conformation in water: Molecular dynamics and experimental characterization2005In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 89, no 2, 842-857 p.Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics simulations of a ribonuclease A C-peptide analog and a sequence variant were performed in water at 277 and 300 K and in 8 M urea to clarify the molecular denaturation mechanism induced by urea and the early events in protein unfolding. Spectroscopic characterization of the peptides showed that the C-peptide analog had a high alpha-helical content, which was not the case for the variant. In the simulations, interdependent side-chain interactions were responsible for the high stability of the alpha-helical C-peptide analog in the different solvents. The other peptide displayed alpha-helical unwinding that propagated cooperatively toward the N-terminal. The conformations sampled by the peptides depended on their sequence and on the solvent. The ability of water molecules to form hydrogen bonds to the peptide as well as the hydrogen bond lifetimes increased in the presence of urea, whereas water mobility was reduced near the peptide. Urea accumulated in excess around the peptide, to which it formed long-lived hydrogen bonds. The unfolding mechanisms induced by thermal denaturation and by urea are of a different nature, with urea-aqueous solutions providing a better peptide solvation than pure water. Our results suggest that the effect of urea on the chemical denaturation process involves both the direct and indirect mechanisms.

  • 10. Chin, T M
    et al.
    Berndt, Kurt D
    Universtity of Chicago, USA.
    Yang, N C C
    Self-Assembling Hexameric Helical Bundle Forming Peptides1992In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 114, no 6, 2279-2280 p.Article in journal (Refereed)
  • 11.
    Elgan, Tobias H.
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences. Karolinska Insitute.
    Quantifying Escherichia coli Glutaredoxin-3 Substrate Specificity Using Ligand-induced Stability2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 47, 32839-32847 p.Article in journal (Refereed)
    Abstract [en]

    Traditionally, quantification of protein-ligand affinity is performed using kinetic or equilibrium measurements. However, if the binding reaction proceeds via a stable covalent complex, these approaches are often limited. By exploiting the fact that the conformational stabilization of a protein is altered upon ligand binding due to specific interactions, and using an array of selectively chosen ligand analogs, one can quantify the contribution individual interactions have on specificity. We have used ligand-induced stability as a basis to dissect the interaction between glutaredoxin-3 (Grx3) and one of its native substrates, the tripeptide glutathione. Taking advantage of the fact that Grx3 can be trapped in a covalent mixed disulfide to glutathione or to selected synthetic glutathione analogs as part of the natural catalytic cycle, individual contributions to binding of specific molecular groups can be quantified by changes in ligand-induced stability. These changes in conformational stability are interpreted in terms of interaction energies (i.e. specificity) of the particular groups present on the ligand analog. Our results illustrate that although Grx3 recognizes glutathione predominantly through independent and additive ionic interactions at the N- and C-terminal of glutathione, van der Waals interactions from the unique gamma-glutamate moiety of glutathione also play an important role. This study places us closer to understanding the complex task of accommodating multiple substrate specificities in proteins of the thioredoxin superfamily and underscores the general applicability of ligand-induced stability to probe substrate specificity.

  • 12.
    Elgán, Tobias H.
    et al.
    Södertörn University, School of Life Sciences. Karlolinska instituet.
    Planson, A. -G
    Beckwith, J.
    Güntert, P.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry. Karolinska institutet.
    Determinants of activity in glutaredoxins: An in vitro evolved Grx1-like variant of Escherichia coli Grx32010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 430, no 3, 487-495 p.Article in journal (Refereed)
    Abstract [en]

    The Escherichia coli glutaredoxins 1 and 3 (Grx1 and Grx3) are structurally similar (37% sequence identity), yet have different activities in vivo. Unlike Grx3, Grx1 efficiently reduces protein disulfides in proteins such as RR (ribonucleotide reductase), whereas it is poor at reducing S-glutathionylated proteins. An E. coli strain lacking genes encoding thioredoxins 1 and 2 and Grx1 is not viable on either rich or minimal medium; however, a M43V mutation in Grx3 restores growth under these conditions and results in a Grx1-like protein [Ortenberg, Gon, Porat and Beckwith (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7439-7944]. To uncover the structural basis of this change in activity, we have compared wild-type and mutant Grx3 using CD and NMR spectroscopy. Ligand-induced stability measurements demonstrate that the Grx3(M43V/C65Y) mutant has acquired affinity for RR. Far-UV CD spectra reveal no significant differences, but differences are observed in the near-UV region indicative of tertiary structural changes. NMR 1H- 15N HSQC (heteronuclear single quantum coherence) spectra show that approximately half of the 82 residues experience significant (Δδ > 0.03 p.p.m.) chemical shift deviations in the mutant, including nine residues experiencing extensive (Δδ ≥ 0.15 p.p.m.) deviations. To test whether the M43V mutation alters dynamic properties of Grx3, H/D (hydrogen/deuterium) exchange experiments were performed demonstrating that the rate at which backbone amides exchange protons with the solvent is dramatically enhanced in the mutant, particularly in the core of the protein. These data suggest that the Grx1-like activity of the Grx3(M43V/C65Y) mutant may be explained by enhanced intrinsic motion allowing for increased specificity towards larger substrates such as RR.

  • 13.
    Ferreira, Monica E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry.
    Nilsson, Johan
    Södertörn University, School of Life Sciences, Molecular biology.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology.
    WD40 Domain Divergence Is Important for Functional Differences between he Fission Yeast Tup11 and Tup12 Co-Repressor Proteins2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 6, e11009- p.Article in journal (Refereed)
    Abstract [en]

    We have previously demonstrated that subsets of Ssn6/Tup target genes ave distinct requirements for the Schizosaccharomyces pombe homologs of he Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very igh level of divergence in the histone interacting repression domains f the two proteins suggested that determinants distinguishing Tup11 and up12 might be located in this domain. Here we have combined hylogenetic and structural analysis as well as phenotypic haracterization, under stress conditions that specifically require up12, to identify and characterize the domains involved in up12-specific action. The results indicate that divergence in the epression domain is not generally relevant for Tup12-specific function. nstead, we show that the more highly conserved C-terminal WD40 repeat omain of Tup12 is important for Tup12-specific function. Surface amino cid residues specific for the WD40 repeat domain of Tup12 proteins in ifferent fission yeasts are clustered in blade 3 of the propeller-like tructure that is characteristic of WD40 repeat domains. The Tup11 and up12 proteins in fission yeasts thus provide an excellent model system or studying the functional divergence of WD40 repeat domains.

  • 14.
    Ferreira, Monica E
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Hermann, Stefan
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Prochasson, P
    Stowers Institute for Medical Research, Kansas City, USA.
    Workman, J L
    Stowers Institute for Medical Research, Kansas City, USA.
    Berndt, Kurt D
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Athony P H
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Mechanism of transcription factor recruitment by acidic activators2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 23, 21779-21784 p.Article in journal (Refereed)
    Abstract [en]

    Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.

  • 15.
    Ferreira, Monica E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Nilsson, Johan
    Södertörn University, School of Life Sciences, Molecular biology.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Protein domains underlying functional divergence between the Tup11 and Tup12 co-repressor proteins in fission yeastManuscript (preprint) (Other academic)
  • 16.
    Ferreira, Monica E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska instiutet.
    Prochasson, Philippe
     Stowers Institute for Medical Research, Kansas City, MO, USA.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry. Karolinska institutet.
    Workman, Jerry L.
    Stowers Institute for Medical Research, Kansas City, MO, USA.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska institutet.
    Activator-binding domains of the SWI/SNF chromatin remodeling complex characterized in vitro are required for its recruitment to promoters in vivo2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 9, 2557-2565 p.Article in journal (Refereed)
    Abstract [en]

    Interaction between acidic activation domains and the activator-binding domains of Swi1 and Snf5 of the yeast SWI/SNF chromatin remodeling complex has previously been characterized in vitro. Although deletion of both activator-binding domains leads to phenotypes that differ from the wild-type, their relative importance for SWI/SNF recruitment to target genes has not been investigated. In the present study, we used chromatin immunoprecipitation assays to investigate the individual and collective importance of the activator-binding domains for SWI/SNF recruitment to genes within the GAL regulon in vivo. We also investigated the consequences of defective SWI/SNF recruitment for target gene activation. We demonstrate that deletion of both activator-binding domains essentially abolishes galactose-induced SWI/SNF recruitment and causes a reduction in transcriptional activation similar in magnitude to that associated with a complete loss of SWI/SNF activity. The activator-binding domains in Swi1 and Snf5 make approximately equal contributions to the recruitment of SWI/SNF to each of the genes studied. The requirement for SWI/SNF recruitment correlates with GAL genes that are highly and rapidly induced by galactose.

  • 17. Filling, C
    et al.
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Intitute.
    Benach, J
    Knapp, S
    Prozorovski, T
    Nordling, E
    Ladenstein, R
    Jörnvall, H
    Oppermann, U
    Critical residues for structure and catalysis in short-chain dehydrogenases/reductases2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 28, 25677-25684 p.Article in journal (Refereed)
    Abstract [en]

    Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wildtype enzyme at 1.2-Angstrom resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.

  • 18. Filling, C
    et al.
    Nordling, E
    Benach, J
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Ladenstein, R
    Jörnvall, H
    Oppermann, U
    Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold2001In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 289, no 3, 712-717 p.Article in journal (Refereed)
    Abstract [en]

    Short-chain dehydrogenases/reductases (SDR) constitute a large family of enzymes found in all forms of life. Despite a low level of sequence identity, the three-dimensional structures determined display a nearly superimposable alpha/beta folding pattern. We identified a conserved asparagine residue located within strand betaF and analyzed its role in the short-chain dehydrogenase/reductase architecture. Mutagenetic replacement of Asn179 by Ala in bacterial 3 beta /17 beta -hydroxysteroid dehydrogenase yields a folded, but enzymatically inactive enzyme, which is significantly more resistant to denaturation by guanidinium hydrochloride. Crystallographic analysis of the wild-type enzyme at 1.2-Angstrom resolution reveals a hydrogen bonding network, including a buried and well-ordered water molecule connecting strands betaE to betaF, a common feature found in 16 of 21 known three-dimensional structures of the family. Based on these results, we hypothesize that in mammalian 11 beta -hydroxysteroid dehydrogenase the essential Asn-linked glycosylation site, which corresponds to the conserved segment, displays similar structural features and has a central role to maintain the SDR scaffold.

  • 19. Foloppe, N
    et al.
    Sagemark, Johan
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Nordstrand, K
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Nilsson, L
    Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: Comparison with functionally related proteins2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 310, no 2, 449-470 p.Article in journal (Refereed)
    Abstract [en]

    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.

  • 20. Güntert, P
    et al.
    Berndt, Kurt D
    Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Wüthrich, K
    The Program Asno for Computer-Supported Collection of Noe Upper Distance Constraints as Input for Protein-Structure Determination1993In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 3, no 5, 601-606 p.Article in journal (Refereed)
    Abstract [en]

    A new program, ASNO ('ASsign NOes'), for computer-supported NOE cross-peak assignments is described. ASNO is used for structure refinement in several rounds of NOESY cross-peak assignments and 3D structure calculations, where the preliminary structures are used as a reference to resolve ambiguities in NOE assignments which are otherwise based on the chemical shifts available from the sequence-specific resonance assignments. The practical use of ASNO for proteins is illustrated with the structure determination of Dendrotoxin K from Dendroaspis polylepis polylepis.

  • 21. Hammarström, A
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Sillard, R
    Adermann, K
    Otting, G
    Solution structure of a naturally-occurring zinc-peptide complex demonstrates that the N-terminal zinc-binding module of the Lasp-1 LIM domain is an independent folding unit1996In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 35, no 39, 12723-12732 p.Article in journal (Refereed)
    Abstract [en]

    The three-dimensional solution structure of the 1:1 complex between the synthetic peptide ZF-1 and zinc was determined by H-1 NMR spectroscopy. The peptide, initially isolated from pig intestines, is identical in sequence to the 30 N-terminal amino acid residues of the human protein Lasp-1 belonging to the LIM domain protein family. The final set of 20 energy-refined NMR conformers has an average rmsd relative to the mean structure of 0.55 Angstrom for the backbone atoms of residues 3-30, Calculations without zinc atom constraints unambiguously identified Cys 5, Cys 8, His 26, and Cys 29 as the zinc-coordinating residues. LIM domains consist of two sequential zinc-binding modules and the NMR structure of the ZF-1(-)zinc complex is the first example of a structure of an isolated module. Comparison with the known structures of the N-terminal zinc-binding modules of both the second LIM domain of chicken CRP and rat GRIP with which ZF-1 shares 50% and 43% sequence identity, respectively, supports the notion that the zinc-binding modules of the LIM domain have a conserved structural motif and identifies local regions of structural diversity. The similarities include conserved zinc-coordinating residues, a rubredoxin knuckle involving Cys 5 and Cys 8, and the coordination of the zinc ion by histidine N-delta in contrast to the more usual coordination by N-epsilon observed for other zinc-finger domains, The present structure determination of the ZF-1(-)zinc complex establishes the N-terminal half of a LIM domain as an independent folding unit. The structural similarities of N- and C-terminal zinc-binding modules of the LIM domains, despite limited sequence identity, lead to the proposal of a single zinc-binding motif in LIM domains. The coordinates are available from the Brookhaven protein data bank, entry 1ZFO.

  • 22.
    Hermann, Stefan
    et al.
    Södertörn University, Avdelning Naturvetenskap.
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap.
    How transcriptional activators bind target proteins2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 43, 40127-40132 p.Article in journal (Refereed)
    Abstract [en]

    The product of the proto-oncogene c-myc influences many cellular processes through the regulation of specific target genes. Through its transactivation domain (TAD), c-Myc protein interacts with several transcription factors, including TATA-binding protein (TBP). We present data that suggest that in contrast to some other transcriptional activators, an extended length of the c-Myc TAD is required for its binding to TBP. Our data also show that this interaction is a multistep process, in which a rapidly forming low affinity complex slowly converts to a more stable form. The initial complex formation results from ionic or polar interactions, whereas the slow conversion to a more stable form is hydrophobic in nature. Based on our results, we suggest two alternative models for activation domain/target protein interactions, which together provide a single universal paradigm for understanding activator-target factor interactions.

  • 23. Holmgren, A
    et al.
    Arnér, E S J
    Berndt, Kurt D
    Karolinska Institutet.
    Glutaredoxin2002In: Encyclopedia of Molecular Biology / [ed] Thomas E. Creighton, John Wiley & Sons, 2002Chapter in book (Other academic)
  • 24. Holmgren, A
    et al.
    Arnér, E S J
    Berndt, Kurt D
    Karolinska Institutet.
    Thioredoxin2002In: Encyclopedia of Molecular Biology / [ed] Thomas E. Creighton, John Wiley & Sons, 2002Chapter in book (Other academic)
  • 25. Holmgren, A
    et al.
    Arnér, E S J
    Berndt, Kurt D
    Karolinska Institutet.
    Thioredoxin Reductase2002In: Encyclopedia of Molecular Biology / [ed] Thomas E. Creighton, John Wiley & Sons, 2002Chapter in book (Other academic)
  • 26. Hurme, R
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Namork, E
    Rhen, M
    DNA binding exerted by a bacterial gene regulator with an extensive coiled-coil domain1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 29, 12626-12631 p.Article in journal (Refereed)
    Abstract [en]

    Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel, sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.

  • 27. Hurme, R
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Normark, S J
    Rhen, M
    A proteinaceous gene regulatory thermometer in Salmonella1997In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 90, no 1, 55-64 p.Article in journal (Refereed)
    Abstract [en]

    Novel utilization of the coiled-coil motif is presented that enables TlpA, an autoregulatory repressor protein in Salmonella, to sense temperature shifts directly and thereby to modulate the extent of transcription repression. Salmonella cells shifted to higher temperatures, such as those encountered at host entry, showed derepressed tlpA activity. tlpA::lacZ fusions indicated that the promoter itself is insensitive to thermal shifts and that transcription control was exerted by the autorepressor TlpA only. In vitro studies with highly purified TlpA showed concentration and temperature dependence for both fully folded conformation and function, indicating that the thermosensing in TlpA is based on monomer-to-coiled-coil equilibrium.

  • 28. Jimenez, A
    et al.
    Johansson, C
    Ljung, J
    Sagemark, Johan
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Ren, B
    Tibbelin, G
    Ladenstein, R
    Kieselbach, T
    Holmgren, A
    Gustafsson, J A
    Miranda-Vizuete, A
    Human spermatid-specific thioredoxin-1 (Sptrx-1) is a two-domain protein with oxidizing activity2002In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 530, no 1-3, 79-84 p.Article in journal (Refereed)
    Abstract [en]

    Spermatid-specific thioredoxin-1 (Sptrx-1) is the first member of the thioredoxin family of proteins with a tissue-specific expression pattern, found exclusively in the tail of elongating spermatids and spermatozoa. We describe here further biochemical characterization of human Sptrx-1 protein structure and enzymatic activity. In gel filtration chromatography human Sptrx-1 eluates as a 400 kDa protein consistent with either an oligomeric form, not maintained by intermolecular disulfide bonding, and/or a highly asymmetrical structure. Analysis of circular dichroism spectra of fragments 1-360 and 361-469 and comparison to spectra of full-length Sptrx-1 supports a two-domain organization with a largely unstructured N-terminal domain and a folded thioredoxin-like C-terminal domain. Functionally, Sptrx-1 behaves as an oxidant in vitro when using selenite, but not oxidized glutathione, as electron acceptor. This oxidizing enzymatic activity suggests that Sptrx-1 might govern the stabilization (by disulfide cross-linking) of the different structures in the developing tail of spermatids and spermatozoa.

  • 29. Johansson, J
    et al.
    Gudmundsson, G H
    Rottenberg, M E
    Berndt, Kurt D
    Karolinska Institutet.
    Agerberth, B
    Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-371998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 6, 3718-3724 p.Article in journal (Refereed)
    Abstract [en]

    The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO3-, SO42-, or CF3CO2- causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl- is less efficient, A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer, The extent of alpha-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL 37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered, In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 mu M, and at 13-25 mu M the peptide is cytotoxic against several eukaryotic cells, In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.

  • 30. Kaiser, L
    et al.
    Velickovic, T C
    Badia-Martinez, D
    Adedoyin, J
    Thunberg, S
    Hallen, D
    Berndt, Kurt D
    Karolinska Institutet.
    Gronlund, H
    Gafvelin, G
    van Hage, M
    Achour, A
    Structural characterization of the tetrameric form of the major cat allergen Fel d 12007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 370, no 4, 714-727 p.Article in journal (Refereed)
    Abstract [en]

    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.

  • 31. Kaumaya, P T P
    et al.
    Berndt, Kurt D
    University of Chicago, USA.
    Heidorn, D B
    Trewhella, J
    Kezdy, F J
    Goldberg, E
    Synthesis and Biophysical Characterization of Engineered Topographic Immunogenic Determinants with Alpha-Alpha-Topology1990In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 29, no 1, 13-23 p.Article in journal (Refereed)
  • 32. Knapp, S
    et al.
    Karshikoff, A
    Berndt, Kurt D
    Karolinska Institutet.
    Christova, P
    Atanasov, B
    Ladenstein, R
    Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus1996In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 264, no 5, 1132-1144 p.Article in journal (Refereed)
    Abstract [en]

    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

  • 33. Knapp, S
    et al.
    Mattson, P T
    Christova, P
    Berndt, Kurt D
    Karolinska Institutet.
    Karshikoff, A
    Vihinen, M
    Smith, C I E
    Ladenstein, R
    Thermal unfolding of small proteins with SH3 domain folding pattern1998In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 31, no 3, 309-319 p.Article in journal (Refereed)
    Abstract [en]

    The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy, The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec), At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. (C) 1998 Wiley-Liss, Inc.

  • 34. Lebbink, J H G
    et al.
    Consalvi, V
    Chiaraluce, R
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Ladenstein, R
    Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: Cooperativity and electrostatic contribution to stability2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 52, 15524-15535 p.Article in journal (Refereed)
    Abstract [en]

    Cooperative interactions within ion-pair networks of hyperthermostable proteins are thought to be a major determinant for extreme protein stability. While the favorable thermodynamic contributions of optimized electrostatics in general as well as those of pairwise interactions have been documented, cooperativity between pairwise interactions has not yet been studied thermodynamically in proteins from hyperthermophiles. In this study we use the isolated cofactor binding domain of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima to analyze pairwise and cooperative interactions within the salt-bridge triad Arg190-Glu231-Lys193. The X-ray structure of the domain was solved at 1.43 Angstrom and reveals the salt-bridge network with surrounding solvent molecules in detail. All three participating charges in the network were mutated to alanine in all combinations. The X-ray structure of the variant lacking all three charges reveals that the removal of the side chains has no effect on the overall conformation of the protein. Using solvent denaturation and thermodynamic cycles, the interaction energies between each pair of residues in the network were determined in the presence and in the absence of the third residue. Both the Arg190-Glu231 ion pair and the Lys193-Glu231 salt bridge in the absence of the third residue, contribute favorably to the free energy for unfolding of the domain in urea. Using guanidinium chloride as denaturant reveals a strong cooperativity between the two ion-pair interactions, the presence of the second ion pair converts the first interaction from destabilizing into stabilizing by as much as 1.09 kcal/mol. The different energetics of the salt-bridge triad in urea and GdmCl are discussed with reference to the observed anion binding in the crystal structure at high ionic strength and their possible role in a highly charged, high-temperature environment such as the cytoplasm of hyperthermophiles.

  • 35. Lerner, S A
    et al.
    Dudek, E J
    Boisvert, W E
    Berndt, Kurt D
    Effect of highly potent antipseudomonal beta-lactam agents alone and in combination with aminoglycosides against Pseudomonas aeruginosa1984In: Reviews Of Infectious Diseases, ISSN 0162-0886, Vol. 6, no Supplement 3, S678-88 p.Article in journal (Refereed)
    Abstract [en]

    The activities of ticarcillin, cefsulodin, ceftazidime, aztreonam, and imipenem, formerly known as N-formimidoyl thienamycin, were evaluated alone and in combination with aminoglycosides against 56 clinical isolates of Pseudomonas aeruginosa, which were characterized by aminoglycoside susceptibility and content of aminoglycoside-modifying enzymatic activities. All beta-lactam agents were highly active against aminoglycoside-susceptible isolates, and with few exceptions the aminoglycoside-resistant isolates were susceptible to all of the beta-lactam agents except ticarcillin. Combinations of the beta-lactam agents with aminoglycosides frequently acted synergistically, but the effect of different beta-lactam agents in combination with an aminoglycoside against individual strains was unpredictable. The presence or absence of an aminoglycoside-modifying enzymatic activity had no observed effect on synergism with tobramycin. Killing-curve experiments with strains in the presence of concentrations of a beta-lactam and an aminoglycoside that were not bactericidal alone (one-fourth the minimal bactericidal concentration) showed synergistic bactericidal action against four strains that were tested. The results demonstrate the great activity of these newer antipseudomonal beta-lactam agents and their potential for synergism with aminoglycosides.

  • 36. Liepinsh, E
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Sillard, R
    Mutt, V
    Otting, G
    Solution Structure and Dynamics of Pec-60, a Protein of the Kazal Type Inhibitor Family, Determined by Nuclear-Magnetic-Resonance Spectroscopy1994In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 239, no 1, 137-153 p.Article in journal (Refereed)
  • 37. Lundholm, L
    et al.
    Bryzgalova, G
    Gao, H
    Portwood, N
    Fält, S
    Berndt, Kurt D
    Karolinska Institutet.
    Dicker, A
    Galuska, D
    Zierath, J R
    Gustafsson, J Å
    Efendic, S
    Dahlman-Wright, K
    Khan, A
    The estrogen receptor {alpha}-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice; potential molecular mechanisms2008In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 199, no 2, 275-286 p.Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

  • 38. Mora, R
    et al.
    Berndt, Kurt D
    University of Chicago, Chicago, USA.
    Tsai, H
    Meredith, S C
    Quantitation of Aspartate and Glutamate in Hplc Analysis of Phenylthiocarbamyl Amino-Acids1988In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 172, no 2, 368-376 p.Article in journal (Refereed)
  • 39. Nilsson, Ola B.
    et al.
    Adedoyin, Justus
    Rhyner, Claudio
    Neimert-Andersson, Theresa
    Grundström, Jeanette
    Berndt, Kurt D
    Karolinska Institutet.
    Crameri, Reto
    Grönlund, Hans
    In vitro evolution of allergy vaccine candidates, with maintained structure, but reduced B cell and T cell activation capacity2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, e24558Article in journal (Refereed)
    Abstract [en]

    Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

  • 40. Nordstrand, K
    et al.
    Sandstrom, A
    Aslund, F
    Holmgren, A
    Otting, G
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap.
    NMR structure of oxidized glutaredoxin 3 from Escherichia coli2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 303, no 3, 423-432 p.Article in journal (Refereed)
    Abstract [en]

    A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.

  • 41. Nordstrand, K
    et al.
    Åslund, F
    Holmgren, A
    Otting, G
    Berndt, Kurt D
    Karolinska Institutet.
    NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: Implications for the enzymatic mechanism1999In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 286, no 2, 541-552 p.Article in journal (Refereed)
    Abstract [en]

    Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family. (C) 1999 Academic Press.

  • 42. Nordstrand, K
    et al.
    Åslund, F
    Meunier, S
    Holmgren, A
    Otting, G
    Berndt, Kurt D
    Karolinska Institutet.
    Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond1999In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 449, no 2-3, 196-200 p.Article in journal (Refereed)
    Abstract [en]

    The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C-11-P-Y-C-14-H-. The H-1 NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cvs-ll thiolate, which is shown to have an abnormally low pK(a) value. A hydrogen bond would also agree,vith activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-ll thiolate, (C) 1999 Federation of European Biochemical Societies.

  • 43. Oppermann, U C T
    et al.
    Filling, C
    Berndt, Kurt D
    Karolinska Institutet.
    Persson, B
    Benach, J
    Ladenstein, R
    Jörnvall, H
    Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 1, 34-40 p.Article in journal (Refereed)
    Abstract [en]

    Mutagenetic replacements uf conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser138 as a member of a catalytically important ''triad'' of residues also involving Tyr151 and Lys155. A Ser --> Ala exchange at position 138 results in an almost complete (>99.9%) loss of enzymatic activity, which is not observed with a Ser --> Thr replacement. This indicates that an essential factor for catalysis is the ability of side chain 138 to form hydrogen bond interactions. Mutations in the NAD(H) binding region, in strands beta A, beta D, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme aad with a differential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3 beta-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the available crystal structure of 3 alpha/20 beta-HSD from Streptomyces hydrogenans suggests a hydrogen her-id in that enzyme between the Thr12 side chain and the backbone NW of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the beta D strand might determine a crucial difference between the reductive and the oxidative reaction types. Similarly, mutation of Asn87 to Ala results in an 80% reduction of K-cat/K-m in the dehydrogenase direction but also unchanged 3-oxoreductase propel ties. It appears that the binding of NAD(+) to the protein is influenced by local structural changes involving strand beta D and beta A to alpha B.

  • 44.
    Sagemark, Johan
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Elgan, Tobias H.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Bürglin, Thomas R.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Johansson, Catrine
    Holmgren, Arne
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Redox properties and evolution of human glutaredoxins2007In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 68, no 4, 879-892 p.Article in journal (Refereed)
    Abstract [en]

    Glutaredoxins (Grxs) are glutathione-dependent oxidoreductases that belong to the thioredoxin superfamily catalyzing thiol-disulfide exchange reactions via active site cysteine residues. Focusing on the human dithiol glutaredoxins having a C-X-Y-C active site sequence motif, the redox potentials of hGrxl and hGrx2 were determined to be -232 and -221 mV, respectively, using a combination of redox buffers, protein-protein equilibrium and thermodynamic linkage. In addition, a nonactive site disulfide was identified between Cys28 and Cys.113 in hGrx2 using redox buffers and chemical digestion. This disulfide confers nearly five kcal mol-1 additional stability by linking the C-terminal helix to the bulk of the protein. The redox potential of this nonactive site disulfide was determined to be -317 mVand is thus expected to be present in all but the most reducing conditions in vivo. As all human glutaredoxins contain additional nonactive site cysteine residues, a full phylogenetic analysis was performed to help elucidate their structural and functional roles. Three distinct groups were found: Grx1, Grx2, and Grx5, the latter representing a highly conserved group of monothiol glutaredoxins having a C-G-F-S active site sequence, with clear homologs from bacteria to human. Grx1 and Grx2 diverged from a common ancestor before the origin vertebrates, possibly even earlier in animal evolution. The highly stabilizing nonactive site disulfide observed in hGrx2 is found to be a conserved feature within the deuterostomes and appears to be the only additional conserved intramolecular disulfide within the glutaredoxins.

  • 45. Tjernberg, L O
    et al.
    Pramanik, A
    Björling, S
    Thyberg, P
    Thyberg, J
    Nordstedt, C
    Berndt, Kurt D
    Karolinska Institutet.
    Terenius, L
    Rigler, R
    Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy1999In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 6, no 1, 53-62 p.Article in journal (Refereed)
    Abstract [en]

    Background: The accumulation of fibrillar deposits of amyloid beta-peptide (A beta) in brain parenchyma and cerebromeningeal blood vessels is a key step in the pathogenesis of Alzheimer's disease. In this report, polymerization of A beta was studied using fluorescence correlation spectroscopy (FCS), a technique capable of detecting small molecules and large aggregates simultaneously in solution. Results: The polymerization of A beta dissolved in Tris-buffered saline, pH 7.4, occurred above a critical concentration of 50 mu M and proceeded from monomers/dimers into two discrete populations of large aggregates, without any detectable amount of oligomers. The aggregation showed very high cooperativity and reached a maximum after 40 min, followed by an increase in the amount of monomers/dimers and a decrease in the size of the large aggregates. Electron micrographs of samples prepared at the time for maximum aggregation showed a mixture of an amorphous network and short diffuse fibrils, whereas only mature amyloid fibrils were detected after one day of incubation. The aggregation was reduced when A beta was incubated in the presence of A beta ligands, oligopeptides previously shown to inhibit fibril formation, and aggregates were partly dissociated after the addition of the ligands. Conclusions: The polymerization of A beta is a highly cooperative process in which the formation of very large aggregates precedes the formation of fibrils. The entire process can be inhibited and, at least in early stages, partly reversed by A beta ligands.

  • 46. Vilhelmsson, Monica
    et al.
    Glaser, Andreas G.
    Martinez, Daniel Badia
    Schmidt, Margit
    Johansson, Catharina
    Rhyner, Claudio
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Scheynius, Annika
    Crameri, Reto
    Achour, Adnane
    Zargari, Arezou
    Mutational analysis of amino acid residues involved in IgE-binding to the Malassezia sympodialis allergen Mala s 112008In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 46, no 2, 294-303 p.Article in journal (Refereed)
    Abstract [en]

    The yeast Malassezia sympodialis, which is an integral part of the normal cutaneous flora, has been shown to elicit specific IgE- and T-cell reactivity in atopic eczema (AE) patients. The M. sympodialis allergen Mala s 11 has a high degree of amino acid sequence homology to manganese superoxide dismutase (MnSOD) from Homo sapiens (50%) and Aspergillus fumigatus (56%). Humoral and cell-mediated cross-reactivity between MnSOD from H. sapiens and A. fumigatus has been demonstrated. Taken together with the recent finding that human MnSOD (hMnSOD) can act as an autoallergen in AE patients sensitised to M. sympodialis, we hypothesized that cross-reactivity could also occur between hMnSOD and Mala s 11, endogenous hMnSOD thus being capable of stimulating an immune response through molecular mimicry. Herein we demonstrate that recombinant Mala s 11 (rMala s 11) is able to inhibit IgE-binding to recombinant hMnSOD and vice versa, indicating that these two homologues share common IgE-binding epitopes and providing an explanation at a molecular level for the autoreactivity to hMnSOD observed in AE patients sensitised to Mala s 11. Using molecular modelling and mapping of identical amino acids exposed on the surface of both Mala s 11 and hMnSCE) we identified four regions each composed of 4-5 residues which are potentially involved in IgE-mediated cross-reactivity. Mutated rMala s 11 molecules were produced in which these residues were altered. Native-like folding was verified by enzymatic activity tests and circular dichroism. The rMala s 11 mutants displayed lower IgE-binding in comparison to wild-type rMala s 11 using plasma from AE patients. In particular, mutation of the residues E29, P30, E122 and K125 lowered the IgE-binding to Mala s 11. The results of this study provide new insights in the molecular basis underlying the cross-reactivity between Mala s 11 and hMnSOD.

  • 47. Wade, D
    et al.
    Palma, M
    Flock, J I
    Berndt, Kurt D
    Karolinska Institutet.
    Silberring, J
    Galaktionov, S G
    Structural analysis of Efb, a fibrinogen binding protein of Staphylococcus aureus1998In: Protein peptide letters, ISSN 0929-8665, E-ISSN 1875-5305, Vol. 5, no 4, 199-206 p.Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus produces and secretes a small, basic protein, designated Efb, that binds to fibrinogen and seems to be required for virulence of the organism. A 3D model of Efb was developed, and it predicts that the C-terminal half of the protein contains substantial alpha-helical content. CD analysis of Efb yielded a value of 40-41% alpha-helix. Calcium and zinc both influence the interactions between Efb and fibrinogen, and an analysis of the amino acid sequence of Efb revealed the presence of consensus sequences for the binding of both metals.

  • 48. Wagner, Claudia S.
    et al.
    Roelle, Alexander
    Cosman, David
    Ljunggren, Hans-Gustaf
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences. Karolinska Intstiute.
    Achour, Adnane
    Structural elements underlying the high binding affinity of human cytomegalovirus UL18 to leukocyte immunoglobulin-like receptor-12007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 373, no 3, 695-705 p.Article in journal (Refereed)
    Abstract [en]

    Human cytomegalovirus (HCMV) encodes UL18, a major histocompatibility complex (MHC) class I homologue that binds to the leukocyte immunoglobulin-like receptor (LIR)-1 (also called ILT2/CD85j/LILRB1), an inhibitory receptor expressed on myeloid and lymphoid immune cells. The molecular basis underlying the high affinity binding of UL18 to LIR-1, compared to MHC class I molecules (MHC-I), is unclear. Based on a comparative structural analysis of a molecular model of UL18 with the crystal structure of the HLA-A2/LIR-1 complex, we identified three regions in UL18 influencing interaction with LIR-1. Comparison of the relative binding affinities of mutated UL18 proteins to LIR-1 demonstrated the importance of specific residues in each region. Substitution of residues K42/A43 and Q202, localized in the alpha 1 and alpha 3 domains, respectively, reduced binding affinity to LIR-1 nearly by half. The model also suggested the formation of an additional disulfide bridge in the 0 domain of UL18 between residues C240 and C255, not present in MHC-I. Substitution of either cysteine residue prevented association of UL18 to beta(2)m, abolishing binding to LIR-1. All observed differences in binding affinities translated directly into functional consequences in terms of inhibition of IFN-gamma production by T cells, mediated through the UL18-LIR-1 interaction. The larger amount of interacting regions, combined with an increased stability of the alpha 3 and beta(2)m domains allow a higher recognition affinity of UL18 by LIR-1.

  • 49. Wu, X.
    et al.
    Jörnvall, H.
    Berndt, Kurt D.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institutet.
    Oppermann, U.
    Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 313, no 1, 89-96 p.Article in journal (Refereed)
    Abstract [en]

    Expression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.

  • 50. Wu, X
    et al.
    Oppermann, M
    Berndt, Kurt D
    Karolinska Institutet.
    Bergman, T
    Jörnvall, H
    Knapp, S
    Oppermann, U
    Thermal unfolding of the archaeal DNA and RNA binding protein Ssh102008In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 373, no 4, 482-487 p.Article in journal (Refereed)
    Abstract [en]

    The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5-7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 degrees C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of DeltaH(cal) is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (DeltaG (T)) and results in a maximal DeltaG of only 9.5 kcal/mol at 320 K and a DeltaG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.

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