53 results on '"A. Elisabeth Sauer-Eriksson"'
Search Results
2. Structural Basis for GTP versus ATP Selectivity in the NMP Kinase AK3
- Author
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Magnus Wolf-Watz, Christian Hedberg, Christin Grundström, Per Rogne, Kwangho Nam, Jack Goodman, A. Elisabeth Sauer-Eriksson, Marie Rosselin, and Beata Dulko-Smith
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biology ,GTP' ,Kinase ,Adenylate Kinase ,Active site ,Adenylate kinase ,AMP binding ,Molecular Dynamics Simulation ,Biochemistry ,Article ,Substrate Specificity ,Citric acid cycle ,Adenosine Triphosphate ,Structural biology ,Biocatalysis ,biology.protein ,Biophysics ,Humans ,Phosphorylation ,Guanosine Triphosphate ,Protein Binding - Abstract
ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3 which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and non-productively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and non-productive binding by the incorrect substrate bears strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from non-linearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These non-specific interactions likely act to lower the available intra-cellular GTP and ATP concentrations and may have driven evolution to adapt the Michaelis constants of NMP kinases accordingly.
- Published
- 2020
3. Structure-Based Design of Inhibitors Targeting PrfA, the Master Virulence Regulator of Listeria monocytogenes
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Ingeborg van der Lingen, Afshan Begum, Marie Lindgren, Fredrik Almqvist, Christin Grundström, Kristoffer Brännström, Andrew G. Cairns, Jörgen Johansson, Uwe Sauer, Sabine Hansen, Michael N. Hall, Martina Kulén, and A. Elisabeth Sauer-Eriksson
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Regulator ,Virulence ,Chick Embryo ,medicine.disease_cause ,DNA-binding protein ,Virulence factor ,Microbiology ,03 medical and health sciences ,Bacterial Proteins ,Listeria monocytogenes ,Drug Discovery ,medicine ,Transcriptional regulation ,Animals ,Binding site ,Pathogen ,Chemistry ,Anti-Bacterial Agents ,3. Good health ,030104 developmental biology ,Drug Design ,Molecular Medicine ,Peptide Termination Factors - Abstract
Listeria monocytogenes is a bacterial pathogen that controls much of its virulence through the transcriptional regulator PrfA. In this study, we describe structure-guided design and synthesis of a set of PrfA inhibitors based on ring-fused 2-pyridone heterocycles. Our most effective compound decreased virulence factor expression, reduced bacterial uptake into eukaryotic cells, and improved survival of chicken embryos infected with L. monocytogenes compared to previously identified compounds. Crystal structures identified an intraprotein “tunnel” as the main inhibitor binding site (AI), where the compounds participate in an extensive hydrophobic network that restricts the protein’s ability to form functional DNA-binding helix–turn–helix (HTH) motifs. Our studies also revealed a hitherto unsuspected structural plasticity of the HTH motif. In conclusion, we have designed 2-pyridone analogues that function as site-AI selective PrfA inhibitors with potent antivirulence properties.
- Published
- 2018
4. Structure of the <scp>N</scp> ‐terminal domain of the metalloprotease <scp>P</scp> rt <scp>V</scp> from <scp> V </scp> ibrio cholerae
- Author
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B. Göran Karlsson, Sun Nyunt Wai, Cecilia Persson, Anders Öhman, Aaron Edwin, A. Elisabeth Sauer-Eriksson, and Maxim Mayzel
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Coiled coil ,chemistry.chemical_classification ,Proteases ,Metalloproteinase ,Protease ,Stereochemistry ,medicine.medical_treatment ,Active site ,Biology ,medicine.disease_cause ,Biochemistry ,Amino acid ,Microbiology ,chemistry ,Vibrio cholerae ,Hydrolase ,biology.protein ,medicine ,Molecular Biology - Abstract
The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.
- Published
- 2015
5. Principles of ATP and GTP Selectivity in NMP Kinases
- Author
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Christian Hedberg, Magnus Wolf-Watz, Elisabeth Sauer-Eriksson, Uwe Sauer, and Per Rogne
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GTP' ,Chemistry ,Kinase ,Biophysics ,Selectivity - Published
- 2020
6. Interspecies Variation between Fish and Human Transthyretins in Their Binding of Thyroid-Disrupting Chemicals
- Author
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A. Elisabeth Sauer-Eriksson, Christin Grundström, Irina Iakovleva, Patrik L. Andersson, Mikael J. Lindberg, Kristoffer Brännström, Anders Olofsson, and Jin Zhang
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endocrine system ,Thyroid Hormones ,Thyroid Gland ,Endocrine System ,chemistry.chemical_compound ,0404 agricultural biotechnology ,medicine ,Environmental Chemistry ,Endocrine system ,Animals ,Humans ,Prealbumin ,Binding site ,biology ,Thyroid ,nutritional and metabolic diseases ,04 agricultural and veterinary sciences ,General Chemistry ,040401 food science ,Sea Bream ,Transport protein ,Transthyretin ,medicine.anatomical_structure ,Biochemistry ,chemistry ,biology.protein ,Xenobiotic ,Homeostasis ,Hormone - Abstract
Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin (TTR) is found in the serum and cerebrospinal fluid of vertebrates, where it transports thyroid hormones. Here, we explored the interspecies variation in TDC binding to human and fish TTR (exemplified by Gilthead seabream ( Sparus aurata)). The in vitro binding experiments showed that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR, in particular, the polar TDCs (500-fold lower affinity). Crystal structures of the seabream TTR-TDC complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding deep in the hormone binding cavity, which explains their low affinity to seabream TTR. Molecular dynamics and in silico alanine scanning simulation also suggested that the protein backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer electrostatic contributions than Ser117 to ligand binding. This provides an explanation for the weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder thyroid-related effects in fish.
- Published
- 2018
7. Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae
- Author
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A. Elisabeth Sauer-Eriksson, Christin Grundström, Aaron Edwin, Sun Nyunt Wai, Gunter Stier, and Anders Öhman
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Proteases ,Recombinant Fusion Proteins ,Genetic Vectors ,Molecular Sequence Data ,Gene Expression ,medicine.disease_cause ,Microbiology ,Metalloprotease ,Fusion tag ,Catalytic Domain ,Escherichia coli ,medicine ,Amino Acid Sequence ,Cloning, Molecular ,Nuclear Magnetic Resonance, Biomolecular ,Vibrio cholerae ,Metalloproteinase ,Nitrogen Isotopes ,biology ,Expression screen ,Kemi ,Isolation (microbiology) ,biology.organism_classification ,Vibrio ,Protein Structure, Tertiary ,Biochemistry ,PrtV ,Proteolysis ,Chemical Sciences ,Sequence Alignment ,Bacteria ,Function (biology) ,Peptide Hydrolases ,Plasmids ,Biotechnology - Abstract
The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria’s ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23–103), the PKD1 domain (residues 755–839), and a 25kDa fragment (residues 581–839). The soluble fusion proteins were captured with Ni2+ affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10–15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on 15N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.
- Published
- 2014
8. Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria
- Author
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Afshan Begum, Jörgen Johansson, Christin Grundström, A. Elisabeth Sauer-Eriksson, Uwe Sauer, Mikael J. Lindberg, Michael N. Hall, and Fredrik Almqvist
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0301 basic medicine ,DNA, Bacterial ,030106 microbiology ,Allosteric regulation ,Amino Acid Motifs ,Glycine ,Biology ,medicine.disease_cause ,Crystallography, X-Ray ,DNA-binding protein ,03 medical and health sciences ,chemistry.chemical_compound ,Listeria monocytogenes ,Bacterial Proteins ,Transcription (biology) ,medicine ,Regulation of gene expression ,Multidisciplinary ,Virulence ,Activator (genetics) ,Gene Expression Regulation, Bacterial ,biochemical phenomena, metabolism, and nutrition ,Biological Sciences ,Glutathione ,Cytosol ,030104 developmental biology ,Biochemistry ,chemistry ,Trans-Activators ,Protein Multimerization ,DNA ,Peptide Termination Factors ,Protein Binding ,Transcription Factors - Abstract
Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.
- Published
- 2016
9. Structure-based design of inhibitors targeting PrfA, the master virulence regulator in Listeria monocytogenes
- Author
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Jörgen Johansson, Christin Grundström, Fredrik Almqvist, Martina Kulén, Elisabeth Sauer-Eriksson, Marie Lindgren, and Uwe Sauer
- Subjects
Regulator ,Virulence ,Biology ,Condensed Matter Physics ,medicine.disease_cause ,Biochemistry ,Microbiology ,Inorganic Chemistry ,Listeria monocytogenes ,Structural Biology ,medicine ,Structure based ,General Materials Science ,Physical and Theoretical Chemistry - Published
- 2018
10. Positive and Negative Substrate Interference Supported by Coinciding Enzyme Residues
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Per Rogne, A. Elisabeth Sauer-Eriksson, Christian Hedberg, Uwe Sauer, and Magnus Wolf-Watz
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chemistry.chemical_classification ,Enzyme ,Interference (communication) ,Chemistry ,Biophysics ,Substrate (chemistry) - Published
- 2019
11. Promiscuous DNA synthesis by human DNA polymerase θ
- Author
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A. Elisabeth Sauer-Eriksson, Matthew Hogg, and Erik Johansson
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0303 health sciences ,DNA clamp ,biology ,Nucleic Acid Enzymes ,Base Pair Mismatch ,DNA polymerase ,Oligonucleotide ,DNA polymerase II ,030302 biochemistry & molecular biology ,Oligonucleotides ,DNA Polymerase Theta ,DNA ,DNA-Directed DNA Polymerase ,Molecular biology ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,Genetics ,biology.protein ,Humans ,DNA Breaks, Double-Stranded ,Primer (molecular biology) ,Polymerase ,030304 developmental biology - Abstract
The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3'-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks.
- Published
- 2011
12. Structure of FocB - a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli
- Author
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Stina Lindberg, Bernt Eric Uhlin, Shenghua Huang, Christin Grundström, Elisabeth Sauer-Eriksson, and Ulrika Wikström Hultdin
- Subjects
Fimbria ,Repressor ,Cell Biology ,Biology ,bacterial infections and mycoses ,urologic and male genital diseases ,Host tissue ,medicine.disease_cause ,Biochemistry ,female genital diseases and pregnancy complications ,Microbiology ,Bacterial adhesin ,Transcription (biology) ,medicine ,bacteria ,Molecular Biology ,Transcription factor ,Escherichia coli - Abstract
In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed in order to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involv ...
- Published
- 2010
13. Quenched hydrogen/deuterium exchange NMR characterization of amyloid-β peptide aggregates formed in the presence of Cu2+or Zn2+
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Malin Lindhagen-Persson, Anders Öhman, A. Elisabeth Sauer-Eriksson, Monika Vestling, and Anders Olofsson
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Programmed cell death ,Magnetic Resonance Spectroscopy ,Hydrogen ,chemistry.chemical_element ,Plaque, Amyloid ,Peptide ,Microscopy, Atomic Force ,Biochemistry ,Alzheimer Disease ,Nephelometry and Turbidimetry ,Divalent metal ions ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Chemistry ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Deuterium ,Peptide Fragments ,Recombinant Proteins ,Amyloid β peptide ,Zinc ,Crystallography ,Biophysics ,Hydrogen–deuterium exchange ,Copper - Abstract
Alzheimer's disease, a neurodegenerative disorder causing synaptic impairment and neuronal cell death, is strongly correlated with aggregation of the amyloid-beta peptide (Abeta). Divalent metal ions such as Cu(2+) and Zn(2+) are known to significantly affect the rate of aggregation and morphology of Abeta assemblies in vitro and are also found at elevated levels within cerebral plaques in vivo. The present investigation characterized the architecture of the aggregated forms of Abeta(1-40) and Abeta(1-42) in the presence or absence of either Cu(2+) or Zn(2+) using quenched hydrogen/deuterium exchange combined with solution NMR spectroscopy. The NMR analyses provide a quantitative and residue-specific structural characterization of metal-induced Abeta aggregates, showing that both the peptide sequence and the type of metal ion exert an impact on the final architecture. Common features among the metal-complexed peptide aggregates are two solvent-protected regions with an intervening minimum centered at Asn27, and a solvent-accessible N-terminal region, Asp1-Lys16. Our results suggest that Abeta in complex with either Cu(2+) or Zn(2+) can attain an aggregation-prone beta-strand-turn-beta-strand motif, similar to the motif found in fibrils, but where the metal binding to the N-terminal region guides the peptide into an assembly distinctly different from the fibril form.
- Published
- 2009
14. Stability and fibril formation properties of human and fish transthyretin, and of the Escherichia coli transthyretin-related protein
- Author
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A. Elisabeth Sauer-Eriksson, Gunilla T. Westermark, Erik Lundberg, and Anders Olofsson
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HIU hydrolase ,Amyloid ,biology ,Chemistry ,macromolecular substances ,Cell Biology ,medicine.disease_cause ,Biochemistry ,Amyloid disease ,Transthyretin ,Fibril formation ,medicine ,biology.protein ,%22">Fish ,sense organs ,Molecular Biology ,Escherichia coli ,Native structure - Abstract
Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease. Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amylo ...
- Published
- 2009
15. Hormone affinity and fibril formation of piscine transthyretin: The role of the N-terminal
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Isabel Morgado, Deborah M. Power, Nídia Estrela, A. Elisabeth Sauer-Eriksson, Eduardo P. Melo, and Erik Lundberg
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Electrophoresis ,Fish Proteins ,Amyloid ,endocrine system ,medicine.medical_specialty ,Recombinant protein ,Ligand binding characteristics ,Transthyretin ,Biochemistry ,Protein Structure, Secondary ,law.invention ,03 medical and health sciences ,Endocrinology ,Fibril formation ,law ,biology.animal ,Internal medicine ,medicine ,Animals ,Prealbumin ,Molecular Biology ,Amyloid fibrils ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Triiodothyronine ,biology ,Protein Stability ,030302 biochemistry & molecular biology ,Life Sciences ,nutritional and metabolic diseases ,Vertebrate ,Amyloid fibril ,Recombinant Proteins ,Sea Bream ,Protein Structure, Tertiary ,Molecular Weight ,Thyroxine ,Thyroid hormones ,TTR tetramer stability ,biology.protein ,Recombinant DNA ,Hormone - Abstract
Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.
- Published
- 2008
16. Interaction of signal-recognition particle 54 GTPase domain and signal-recognition particle RNA in the free signal-recognition particle
- Author
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Shenghua Huang, A. Elisabeth Sauer-Eriksson, and Tobias Hainzl
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Models, Molecular ,Signal peptide ,Archaeal Proteins ,Methanococcus ,Molecular Sequence Data ,GTPase ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,environment and public health ,GTP Phosphohydrolases ,Structure-Activity Relationship ,Protein structure ,RNA, Small Cytoplasmic ,Protein targeting ,medicine ,Signal recognition particle RNA ,Signal recognition particle receptor ,Signal recognition particle ,Binding Sites ,Multidisciplinary ,Base Sequence ,RNA ,Biological Sciences ,Molecular biology ,Protein Structure, Tertiary ,Biophysics ,Nucleic Acid Conformation ,Signal Recognition Particle - Abstract
The signal-recognition particle (SRP) is a ubiquitous protein–RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-Å resolution of the SRP54–SRP19–SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.
- Published
- 2007
17. Heating of proteins as a means of improving crystallization: a successful case study on a highly amyloidogenic triple mutant of human transthyretin
- Author
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Anders Karlsson and A. Elisabeth Sauer-Eriksson
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Amyloid ,Hot Temperature ,Biophysics ,macromolecular substances ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,law.invention ,Genetic Heterogeneity ,Structural Biology ,law ,Genetics ,medicine ,Humans ,Prealbumin ,Crystallization ,Mutation ,biology ,Chemistry ,Condensed Matter Physics ,Transport protein ,Triple mutant ,Transthyretin ,Crystallization Communications ,biology.protein - Abstract
The use of high temperatures in the purification procedures of heat-stable proteins is a well established technique. Recently, rapid pre-heat treatment of protein samples prior to crystallization trials was described as a final polishing step to improve the diffraction properties of crystals [Pusey et al. (2005), Prog. Biophys. Mol. Biol. 88, 359-386]. The present study demonstrates that extended high-temperature incubation (328 K for 48 h) of the highly amyloidogenic transthyretin mutant TTR G53S/E54D/L55S successfully removes heterogeneities and allows the reproducible growth of well diffracting crystals. Heat treatment might be applied as an optimization method to other cases in which the protein/biomolecule fails to form diffracting crystals.
- Published
- 2007
18. Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae
- Author
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Aaron, Edwin, Cecilia, Persson, Maxim, Mayzel, Sun Nyunt, Wai, Anders, Öhman, B Göran, Karlsson, and A Elisabeth, Sauer-Eriksson
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Models, Molecular ,N‐terminal domain ,Protein Structure, Secondary ,NMR ,Protein Structure, Tertiary ,Vibrio cholera ,Catalytic Domain ,PrtV ,metalloproteases ,Protein Structure Reports ,Nuclear Magnetic Resonance, Biomolecular ,Vibrio cholerae ,Conserved Sequence ,Peptide Hydrolases - Abstract
The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N‐terminal domain (residues 23–103) that contains two short α‐helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C‐terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important., Interactive Figure 1 | PDB Code(s): 5abk
- Published
- 2015
19. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state
- Author
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Jörgen Ådén, Michael Kovermann, Uwe Sauer, Magnus Wolf-Watz, A. Elisabeth Sauer-Eriksson, and Christin Grundström
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Protein Conformation ,Biophysics ,General Physics and Astronomy ,Adenylate kinase ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Article ,General Biochemistry, Genetics and Molecular Biology ,Molecular dynamics ,Protein structure ,Catalytic Domain ,Transferase ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Binding Sites ,Multidisciplinary ,Chemistry ,Adenylate Kinase ,Rational design ,Kemi ,General Chemistry ,Adenosine Diphosphate ,Biological sciences ,Enzyme ,Chemical physics ,ddc:540 ,Chemical Sciences ,Mutagenesis, Site-Directed ,Ground state ,Protein Binding - Abstract
An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions., Adenylate kinase (AdK) plays a key role in cellular energy homeostasis by catalysing the reversible magnesium-dependent formation of ADP from AMP and ATP. Here the authors present a detailed analysis of adenylate kinase's conformational dynamics and characterize a high-energy state of AdK indispensable for catalysis.
- Published
- 2015
20. The transthyretin-related protein: Structural investigation of a novel protein family
- Author
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Stefan Bäckström, A. Elisabeth Sauer-Eriksson, Erik Lundberg, and Uwe Sauer
- Subjects
Bromides ,Models, Molecular ,Molecular Sequence Data ,chemistry.chemical_element ,Zinc ,Crystallography, X-Ray ,Ligands ,Protein Structure, Secondary ,Protein structure ,Tetramer ,Structural Biology ,Humans ,Prealbumin ,Amino Acid Sequence ,Binding site ,Peptide sequence ,chemistry.chemical_classification ,Binding Sites ,Sequence Homology, Amino Acid ,Chemistry ,Ligand ,Escherichia coli Proteins ,Amino acid ,Transport protein ,Crystallography ,Zinc Compounds ,Multigene Family - Abstract
The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.
- Published
- 2006
21. The Solvent Protection of Alzheimer Amyloid-β-(1–42) Fibrils as Determined by Solution NMR Spectroscopy
- Author
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A. Elisabeth Sauer-Eriksson, Anders Öhman, and Anders Olofsson
- Subjects
Amyloid ,Amyloid β ,Peptide ,Microscopy, Atomic Force ,Fibril ,Biochemistry ,Nuclear magnetic resonance ,mental disorders ,medicine ,Humans ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Chemistry ,Deuterium Exchange Measurement ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,medicine.disease ,Peptide Fragments ,Solvent ,Solubility ,Solvents ,Biophysics ,Alzheimer's disease - Abstract
Alzheimer disease is a neurodegenerative disorder that is tightly linked to the self-assembly and amyloid formation of the 39-43-residue-long amyloid-beta (Abeta) peptide. Considerable evidence suggests a correlation between Alzheimer disease development and the longer variants of the peptide, Abeta-(1-42/43). Currently, a molecular understanding for this behavior is lacking. In the present study, we have investigated the hydrogen/deuterium exchange of Abeta-(1-42) fibrils under physiological conditions, using solution NMR spectroscopy. The obtained residue-specific and quantitative map of the solvent protection within the Abeta-(1-42) fibril shows that there are two protected core regions, Glu11-Gly25 and Lys28-Ala42, and that the residues in between, Ser26 and Asn27, as well as those in the N terminus, Asp1-Tyr10, are solvent-accessible. This result reveals considerable discrepancies when compared with a previous investigation on Abeta-(1-40) fibrils and suggests that the additional residues in Abeta-(1-42), Ile41 and Ala42, significantly increase the solvent protection and stability of the C-terminal region Lys28-Ala42. Consequently, our findings provide a molecular explanation for the increased amyloidogenicity and toxicity of Abeta-(1-42) compared with shorter Abeta variants found in vivo.
- Published
- 2006
22. Cys114-Linked Dimers of Transthyretin Are Compatible with Amyloid Formation
- Author
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A. Elisabeth Sauer-Eriksson, Anders Olofsson, Anders Karlsson, and Therese Eneqvist
- Subjects
Amyloid ,Amyloid Neuropathies, Familial ,biology ,Chemistry ,Stereochemistry ,Dimer ,Intermolecular force ,Sequence (biology) ,Microscopy, Atomic Force ,Fibril ,Biochemistry ,chemistry.chemical_compound ,Transthyretin ,Amino Acid Substitution ,biology.protein ,Humans ,Prealbumin ,Molecule ,Cysteine ,Dimerization - Abstract
The Tyr114Cys substitution in the human plasma protein transthyretin leads to a particularly aggressive form of familial amyloidotic polyneuropathy. In a previous study we demonstrated that ATTR Tyr114Cys forms intermolecular disulfide bonds, which partly impair fibril formation and result in a more amorphous morphology. Apart from the introduced cysteinyl group in position 114, the native sequence contains one cysteine located at position 10. To deduce the role of intermolecular disulfide bridging in fibril formation we generated and characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular cysteine bridge at position 114 enhances the exposure of cysteine 10, thereby facilitating additional intermolecular cysteine assemblies. We also purified a disulfide-linked dimeric form of TTR Cys10Ala/Tyr114Cys, which was recognized by the anti-TTR amyloid-specific monoclonal antibody MAb (39-44). Moreover, this dimeric molecule can form protofibrils indistinguishable from the fibrils formed under reducing conditions, as judged by atomic force microscopy. Assuming that both molecules of the dimer are part of the core of the fibril, the assembly is incompatible with a preserved native or near-native dimeric interphase. Our findings raise the question of whether TTR-amyloid architecture is indeed the result of one highly stringent assembly of structures or if different fibrils may be built from different underlying structures.
- Published
- 2005
23. The Effect of Iodide and Chloride on Transthyretin Structure and Stability
- Author
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Elisabeth Sauer-Eriksson, Anders Olofsson, Ulrika Wikström Hultdin, and Andreas Hörnberg
- Subjects
Models, Molecular ,Protein Denaturation ,Protein Conformation ,Inorganic chemistry ,Iodide ,Halide ,Biochemistry ,Protein structure ,Chlorides ,Tetramer ,Humans ,Prealbumin ,Urea ,chemistry.chemical_classification ,biology ,Anomalous scattering ,nutritional and metabolic diseases ,Hydrogen-Ion Concentration ,Iodides ,Ligand (biochemistry) ,Small molecule ,Crystallography ,Transthyretin ,chemistry ,biology.protein ,Crystallization - Abstract
Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.
- Published
- 2005
24. Structural insights into SRP RNA: An induced fit mechanism for SRP assembly
- Author
-
Tobias Hainzl, Shenghua Huang, and A. Elisabeth Sauer-Eriksson
- Subjects
Models, Molecular ,Conformational change ,Adenosine ,Methanococcus ,Molecular Sequence Data ,Electrophoretic Mobility Shift Assay ,RNA, Archaeal ,Biology ,Crystallography, X-Ray ,Models, Biological ,Article ,Protein Structure, Secondary ,Cytosine ,Protein structure ,Signal recognition particle RNA ,Binding site ,Molecular Biology ,Protein secondary structure ,Signal recognition particle ,Binding Sites ,Base Sequence ,RNA ,Protein Structure, Tertiary ,Lactococcus lactis ,Biochemistry ,Helix ,Chromatography, Gel ,Biophysics ,Nucleic Acid Conformation ,Signal Recognition Particle ,Protein Binding - Abstract
Proper assembly of large protein–RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein–RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-Å resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.
- Published
- 2005
25. High Resolution Crystal Structures of Piscine Transthyretin Reveal Different Binding Modes for Triiodothyronine and Thyroxine
- Author
-
Erik Lundberg, Deborah M. Power, Anders Karlsson, Shenghua Huang, A. Elisabeth Sauer-Eriksson, Therese Eneqvist, and Cecília R.A. Santos
- Subjects
Models, Molecular ,endocrine system ,Extracellular transport ,Protein Conformation ,High resolution ,Crystal structure ,Plasma protein binding ,Crystallography, X-Ray ,Ligands ,Transthyretin ,Biochemistry ,Protein structure ,Escherichia coli ,Animals ,Humans ,Prealbumin ,Cloning, Molecular ,Molecular Biology ,Triiodothyronine ,biology ,nutritional and metabolic diseases ,Cell Biology ,Molecular biology ,Sea Bream ,Protein Structure, Tertiary ,Transport protein ,Thyroxine ,biology.protein ,Chickens ,Protein Binding - Abstract
Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3 -triiodo- L-thyronine (T3) and 3,5,3 ,5 -tetraiodo-L-thyronine (thyroxine, T4). Human TTR has higher affinity for T4 than T3, whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 Å resolution and bound to ligands T3 and T4, both at 1.9 Å resolution. The apo structure is similar to human TTR with structural changes only at -strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR T4 complex shows the T4-binding site to be similar but not identical to human TTR, whereas the TTR T3 complex shows the I3 halogen situated at the site normally occupied by the hydroxyl group of T4. The significantly wider entrance of the hormone- binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T3 and T4. We thank Anders Olofsson, Uwe H. Sauer, Andreas Ho¨rnberg, and Terese Bergfors for valuable discussions and critical reading of the manuscript.
- Published
- 2004
26. S-domain assembly of the signal recognition particle
- Author
-
Tobias Hainzl and A. Elisabeth Sauer-Eriksson
- Subjects
Models, Molecular ,Signal peptide ,Macromolecular Substances ,Protein Conformation ,Biology ,environment and public health ,Ribosome ,Protein Structure, Secondary ,Structure-Activity Relationship ,Protein structure ,Species Specificity ,Structural Biology ,RNA, Small Cytoplasmic ,Escherichia coli ,Humans ,Signal recognition particle RNA ,Molecular Biology ,Signal recognition particle receptor ,Ternary complex ,Ribonucleoprotein ,Signal recognition particle ,Binding Sites ,Crystallography ,Protein Structure, Tertiary ,Biochemistry ,Biophysics ,Signal Recognition Particle ,Protein Binding - Abstract
The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes. In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA. The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.
- Published
- 2003
27. The transthyretin-related protein family
- Author
-
Therese Eneqvist, A. Elisabeth Sauer-Eriksson, Ruben Abagyan, Erik Lundberg, and Lars Nilsson
- Subjects
Protein Folding ,Thyroid Hormones ,DNA, Complementary ,Protein family ,Protein Conformation ,Sequence analysis ,Molecular Sequence Data ,Polymerase Chain Reaction ,Biochemistry ,Anilino Naphthalenesulfonates ,Escherichia coli ,Animals ,Prealbumin ,Amino Acid Sequence ,Homology modeling ,Cloning, Molecular ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Gene ,Conserved Sequence ,Phylogeny ,Genetics ,Multiple sequence alignment ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,Blotting, Northern ,biology.organism_classification ,Uric Acid ,Transport protein ,Purines ,RNA Interference ,Homotetramer - Abstract
A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.
- Published
- 2003
28. Structure of the SRP19–RNA complex and implications for signal recognition particle assembly
- Author
-
Shenghua Huang, A. Elisabeth Sauer-Eriksson, and Tobias Hainzl
- Subjects
Models, Molecular ,Protein Conformation ,Archaeal Proteins ,Methanococcus ,Molecular Sequence Data ,RNA, Archaeal ,Biology ,Crystallography, X-Ray ,environment and public health ,Ribosome ,Tetraloop ,Protein structure ,Humans ,Signal recognition particle RNA ,Amino Acid Sequence ,Signal recognition particle receptor ,Ribonucleoprotein ,Signal recognition particle ,Binding Sites ,Multidisciplinary ,Base Sequence ,RNA ,Eukaryotic Cells ,Biochemistry ,Biophysics ,Nucleic Acid Conformation ,Signal Recognition Particle - Abstract
The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.
- Published
- 2002
29. Organization of an Efficient Carbonic Anhydrase: Implications for the Mechanism Based on Structure−Function Studies of a T199P/C206S Mutant
- Author
-
Björn Sjöblom, Shenghua Huang, § and A. Elisabeth Sauer-Eriksson, and Bengt-Harald Jonsson
- Subjects
Threonine ,Proline ,Stereochemistry ,Carbonic anhydrase II ,Mutant ,Mechanism based ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Substrate Specificity ,Structure-Activity Relationship ,Carbonic anhydrase ,Serine ,Humans ,Cysteine ,Catalytic efficiency ,Carbonic Anhydrases ,Mercaptoethanol ,Binding Sites ,biology ,Chemistry ,Structure function ,Water ,Active site ,Carbon Dioxide ,Lyase ,Recombinant Proteins ,Bicarbonates ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,biology.protein ,Thiocyanates - Abstract
Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.
- Published
- 2002
30. Structural basis of signal-sequence recognition by the signal recognition particle
- Author
-
Gitte Meriläinen, Kristoffer Brännström, Shenghua Huang, Tobias Hainzl, and A. Elisabeth Sauer-Eriksson
- Subjects
Models, Molecular ,Signal peptide ,Signal recognition particle ,Base Sequence ,Basis (linear algebra) ,Archaeal Proteins ,Methanococcus ,Molecular Sequence Data ,Plasma protein binding ,Protein Sorting Signals ,Biology ,Crystallography, X-Ray ,environment and public health ,Ribosome ,Crystallography ,Structural Biology ,Base sequence ,Biological system ,Signal Recognition Particle ,Molecular Biology ,Signal recognition particle receptor ,Protein Binding - Abstract
The signal recognition particle (SRP) recognizes and binds the signal sequence of nascent proteins as they emerge from the ribosome. We present here the 3.0-Å structure of a signal sequence bound to the Methanococcus jannaschii SRP core. Structural comparison with the free SRP core shows that signal-sequence binding induces formation of the GM-linker helix and a 180° flip of the NG domain-structural changes that ensure a hierarchical succession of events during protein targeting.
- Published
- 2011
31. The flavonoid luteolin, but not luteolin-7-O-glucoside, prevents a transthyretin mediated toxic response
- Author
-
Malgorzata Pokrzywa, A. Elisabeth Sauer-Eriksson, Anders Olofsson, Afshan Begum, Malin Walfridsson, and Irina Iakovleva
- Subjects
endocrine system ,Amyloid ,Cell- och molekylärbiologi ,lcsh:Medicine ,Cynaroside ,Flavones ,chemistry.chemical_compound ,Glucosides ,Cell Line, Tumor ,Animals ,Drosophila Proteins ,Humans ,Prealbumin ,Binding site ,lcsh:Science ,Luteolin ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Protein Stability ,lcsh:R ,Wild type ,nutritional and metabolic diseases ,Amyloidosis ,Transport protein ,Transthyretin ,Drosophila melanogaster ,Biochemistry ,chemistry ,biology.protein ,lcsh:Q ,Cell and Molecular Biology ,Research Article - Abstract
Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.
- Published
- 2014
32. The β-Slip
- Author
-
Karin Andersson, A. Elisabeth Sauer-Eriksson, Therese Eneqvist, Anders Olofsson, and Erik Lundgren
- Subjects
biology ,Amyloidosis ,Beta sheet ,Cell Biology ,medicine.disease ,Transthyretin ,Amyloid disease ,Protein structure ,Biochemistry ,Helix ,medicine ,Biophysics ,biology.protein ,Protein folding ,Protein quaternary structure ,Molecular Biology - Abstract
Transthyretin is a tetrameric plasma protein associated with two forms of amyloid disease. The structure of the highly amyloidogenic transthyretin triple mutant TTRG53S/E54D/L55S determined at 2.3 A resolution reveals a novel conformation: the beta-slip. A three-residue shift in beta strand D places Leu-58 at the position normally occupied by Leu-55 now mutated to serine. The beta-slip is best defined in two of the four monomers, where it makes new protein-protein interactions to an area normally involved in complex formation with retinol-binding protein. This interaction creates unique packing arrangements, where two protein helices combine to form a double helix in agreement with fiber diffraction and electron microscopy data. Based on these findings, a novel model for transthyretin amyloid formation is presented.
- Published
- 2000
33. Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide
- Author
-
Bengt-Harald Jonsson, Laura C Chirica, Shenghua Huang, Sven Lindskog, Yafeng Xue, and Elisabeth Sauer-Eriksson
- Subjects
Models, Molecular ,Stereochemistry ,Carbonic anhydrase II ,Molecular Sequence Data ,Crystallography, X-Ray ,Protein Structure, Secondary ,Structural Biology ,Carbonic anhydrase ,Humans ,Molecular replacement ,Amino Acid Sequence ,Disulfides ,Carbonic Anhydrase Inhibitors ,Molecular Biology ,Protein secondary structure ,Carbonic Anhydrases ,Sequence Deletion ,chemistry.chemical_classification ,Binding Sites ,biology ,Active site ,Periplasmic space ,Lyase ,Neisseria gonorrhoeae ,Acetazolamide ,Isoenzymes ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,Sequence Alignment - Abstract
The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.
- Published
- 1998
34. Structural basis for processive DNA synthesis by yeast DNA polymerase ɛ
- Author
-
Else-Britt Lundström, Göran O. Bylund, Matthew Hogg, Pia Osterman, Erik Johansson, Rais Ahmad Ganai, and A. Elisabeth Sauer-Eriksson
- Subjects
DNA Replication ,Models, Molecular ,DNA clamp ,biology ,DNA polymerase ,DNA polymerase II ,DNA replication ,Processivity ,DNA Polymerase II ,Saccharomyces cerevisiae ,DNA polymerase delta ,Biochemistry ,Structural Biology ,Catalytic Domain ,biology.protein ,DNA polymerase I ,Molecular Biology ,DNA polymerase mu - Abstract
DNA polymerase ɛ (Pol ɛ) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ɛ (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ɛ has the highest fidelity among B-family polymerases despite the absence of an extended β-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ɛ to encircle the nascent double-stranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes.
- Published
- 2013
35. Small molecule screening for inhibitors of the YopH phosphatase of Yersinia pseudotuberculosis
- Author
-
Jonas, Eriksson, Christin, Grundström, A Elisabeth, Sauer-Eriksson, Uwe H, Sauer, Hans, Wolf-Watz, and Mikael, Elofsson
- Subjects
Yersinia pseudotuberculosis ,Drug Evaluation, Preclinical ,Protein Tyrosine Phosphatases ,Bacterial Outer Membrane Proteins - Published
- 2012
36. Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis
- Author
-
Uwe Sauer, Mikael Elofsson, A. Elisabeth Sauer-Eriksson, Jonas Eriksson, Hans Wolf-Watz, and Christin Grundström
- Subjects
biology ,Yersinia pestis ,Effector ,Phosphatase ,Virulence ,Yersinia pseudotuberculosis ,Secretion ,Yersinia ,biology.organism_classification ,Yersinia enterocolitica ,Microbiology - Abstract
Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.
- Published
- 2012
37. Assembly and Function of the Signal Recognition Particle from Archaea
- Author
-
Tobias Hainzl, Shenghua Huang, and Elisabeth Sauer-Eriksson
- Subjects
Signal peptide ,Signal recognition particle ,Secretory protein ,Chemistry ,Protein targeting ,medicine ,Signal recognition particle RNA ,GTPase ,medicine.disease_cause ,environment and public health ,Signal recognition particle receptor ,Ribosome ,Cell biology - Abstract
The signal recognition particle (SRP) is a protein-RNA complex that associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. The universally conserved core of SRP consists of SRP RNA and the SRP54 protein, and plays the key role in signal-sequence recognition and binding to the SRP receptor. Critical for SRP function is communication between the two conserved SRP54 domains, the GTPase- and the M-domain, so that signal-sequence binding at the M domain directs receptor binding at the GTPase domain. The structural basis for signal-sequence binding by SRP and subsequent signaling is still poorly understood. By studying the structures of the SRP RNA in its free form as well as in complex with its different protein partners, we have made steady progress towards the elucidation of structural states of the SRP, using the archaeon Methanococcus jannaschii as model system. Together with other structures of SRP proteins and RNA-protein complexes, these structures provide new insights into the mechanisms of SRP-mediated protein targeting.
- Published
- 2011
38. Structure of FocB--a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli
- Author
-
Ulrika W, Hultdin, Stina, Lindberg, Christin, Grundström, Shenghua, Huang, Bernt Eric, Uhlin, and A Elisabeth, Sauer-Eriksson
- Subjects
DNA, Bacterial ,Models, Molecular ,Escherichia coli Proteins ,Amino Acid Motifs ,Molecular Sequence Data ,Membrane Proteins ,Gene Expression Regulation, Bacterial ,Crystallography, X-Ray ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Uropathogenic Escherichia coli ,Amino Acid Sequence ,Adhesins, Bacterial ,Dimerization ,Sequence Alignment ,Transcription Factors - Abstract
In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form homomeric or heteromeric complexes and exert both positive and negative effects on the transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 A resolution. FocB is an all alpha-helical protein with a helix-turn-helix motif. Interestingly, conserved residues important for DNA-binding are located not in the postulated recognition helix of the motif, but in the preceding helix. Results from protein-DNA-binding studies suggest that FocB interacts with the minor groove of its cognate DNA target, which is indicative of a DNA interaction that is unusual for this motif. FocB crystallizes in the form of dimers. Packing interactions in the crystals give two plausible dimerization interfaces. Conserved residues, known to be important for protein oligomerization, are present at both interfaces, suggesting that both sites could play a role in a functional FocB protein.
- Published
- 2010
39. Crystallization and preliminary X-ray diffraction studies of the signal recognition particle receptor FtsY fromMycoplasma mycoides
- Author
-
Talal Gariani and Elisabeth Sauer-Eriksson
- Subjects
Subfamily ,Receptors, Cytoplasmic and Nuclear ,GTPase ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Bacterial Proteins ,Structural Biology ,Escherichia coli ,medicine ,Receptor ,Signal recognition particle receptor ,DNA Primers ,Signal recognition particle ,Base Sequence ,Resolution (electron density) ,Mycoplasma mycoides ,General Medicine ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Biochemistry ,Crystallization ,Signal Recognition Particle - Abstract
The prokaryotic signal recognition particle (SRP) pathway comprises two proteins, Ffh and FtsY, homologous to the SRP54 and SRalpha proteins in the more complex eukaryotic system. All four proteins are part of a unique subfamily of GTPases. Four truncated versions of the 412 amino-acid FtsY receptor protein from Mycoplasma mycoides have been cloned, expressed in Escherichia coli and purified. Purified proteins from all constructs and the full-length FtsY protein were subjected to crystallization trials. Crystals were obtained for the construct which comprised residues 98-412 corresponding to the conserved NG-domain (residues 194-497 in E. coli). A native data set at 1.9 A resolution has been collected at 100 K using synchrotron radiation. The crystals belong to the space group P2(1)2(1)2, with unit-cell parameters a = 68.7, b = 101.1, c = 42.5 A and one molecule in the asymmetric unit.
- Published
- 2000
40. Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli
- Author
-
Gunter Stier, Anders Öhman, Stina Lindberg, Christin Grundström, Anders Allgardsson, Shenghua Huang, Bernt Eric Uhlin, Ulrika Wikström Hultdin, and Elisabeth Sauer-Eriksson
- Subjects
Operon ,Fimbria ,Molecular Sequence Data ,Biophysics ,Biology ,medicine.disease_cause ,Crystallography, X-Ray ,Biochemistry ,Structural Biology ,Transcription (biology) ,Gene cluster ,Genetics ,medicine ,Escherichia coli ,Amino Acid Sequence ,Transcription factor ,Nuclear Magnetic Resonance, Biomolecular ,Adhesins, Escherichia coli ,Circular Dichroism ,Escherichia coli Proteins ,Temperature ,Condensed Matter Physics ,Fusion protein ,Bacterial adhesin ,DNA-Binding Proteins ,Crystallization Communications ,Chromatography, Gel ,Crystallization ,Protein Binding - Abstract
The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His(6)-tagged fusion protein was captured by Ni(2+)-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His(6) tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 A resolution was collected at 100 K using synchrotron radiation.
- Published
- 2009
41. Stability and fibril formation properties of human and fish transthyretin, and of the Escherichia coli transthyretin-related protein
- Author
-
Erik, Lundberg, Anders, Olofsson, Gunilla T, Westermark, and A Elisabeth, Sauer-Eriksson
- Subjects
Amyloid ,Binding Sites ,Escherichia coli Proteins ,Molecular Sequence Data ,Fishes ,Temperature ,Animals ,Humans ,Prealbumin ,Amino Acid Sequence ,Hydrogen-Ion Concentration ,Phylogeny ,Amidohydrolases - Abstract
Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease. Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amyloid fibrils. The transthyretin (TTR)-related proteins comprise a protein family of 5-hydroxyisourate hydrolases with structural similarity to TTR. In this study, we tested the amyloidogenic properties, if any, of sea bream TTR (sbTTR) and Escherichia coli transthyretin-related protein (ecTRP), which share 52% and 30% sequence identity, respectively, with hTTR. We obtained filamentous structures from all three proteins under various conditions, but, interestingly, different structures displayed different tinctorial properties. hTTR and sbTTR formed thin, curved fibrils at low pH (pH 2-3) that bound thioflavin-T (thioflavin-T-positive) but did not stain with Congo Red (CR) (CR-negative). Aggregates formed at the slightly higher pH of 4.0-5.5 had different morphology, displaying predominantly amorphous structures. CR-positive material of hTTR was found in this material, in agreement with previous results. ecTRP remained soluble at pH 2-12 at ambient temperatures. By raising of the temperature, fibril formation could be induced at neutral pH in all three proteins. Most of these temperature-induced fibrils were thicker and straighter than the in vitro fibrils seen at low pH. In other words, the temperature-induced fibrils were more similar to fibrils seen in vivo. The melting temperature of ecTRP was 66.7 degrees C. This is approximately 30 degrees C lower than the melting temperatures of sbTTR and hTTR. Information from the crystal structures was used to identify possible explanations for the reduced thermostability of ecTRP.
- Published
- 2009
42. Transthyretin-Related and Transthyretin-like Proteins
- Author
-
Erik Lundberg, Anna Linusson, and A. Elisabeth Sauer-Eriksson
- Subjects
Genetics ,Transthyretin ,Amyloid ,Protein family ,Structural similarity ,Hydrolase ,biology.protein ,nutritional and metabolic diseases ,Biology ,In vitro ,Function (biology) ,Sequence identity - Abstract
Bioinformatics programs are highly accurate in identifying protein families directly from protein sequences, even when the sequence identity is very low. The transthyretin-related proteins (TRPs) are one example of a protein family that has been identified. Whereas transthyretin (TTR) is well-characterized both in vivo and in vitro, only recently has research focused on the TRPs. Their structural similarity to TTR has been verified, and their function as a 5-hydroxyisourate (HIU) hydrolase has been established. In this review we discuss structural aspects of TRP function. We also discuss the still unknown transthyretin-like proteins (TLPs) that are seemingly unique to nematodes.
- Published
- 2009
43. Amyloid fibril dynamics revealed by combined hydrogen/deuterium exchange and nuclear magnetic resonance
- Author
-
A. Elisabeth Sauer-Eriksson, Anders Olofsson, and Anders Öhman
- Subjects
Amyloid ,Magnetic Resonance Spectroscopy ,Chemistry ,Protein Conformation ,Biophysics ,Deuterium Exchange Measurement ,macromolecular substances ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Fibril ,Biochemistry ,Kinetics ,Protein structure ,Nuclear magnetic resonance ,Solid-state nuclear magnetic resonance ,Hydrogen–deuterium exchange ,Molecular Biology ,Two-dimensional nuclear magnetic resonance spectroscopy - Abstract
A general method to explore the dynamic nature of amyloid fibrils is described, combining hydrogen/deuterium exchange and nuclear magnetic resonance spectroscopy to determine the exchange rates of individual amide protons within an amyloid fibril. Our method was applied to fibrils formed by the amyloid-beta(1-40) peptide, the major protein component of amyloid plaques in Alzheimer's disease. The fastest exchange rates were detected among the first 14 residues of the peptide, a stretch known to be poorly structured within the fibril. Considerably slower exchange rates were observed in the remainder of the peptide within the beta-strand-turn-beta-strand motif that constitutes the fibrillar core.
- Published
- 2008
44. Negatively charged phospholipid membranes induce amyloid formation of medin via an alpha-helical intermediate
- Author
-
Gerhard Gröbner, A. Elisabeth Sauer-Eriksson, Anders Olofsson, and Tomasz Borowik
- Subjects
Circular dichroism ,Amyloid ,Protein Conformation ,Lipid Bilayers ,Phospholipid ,Alpha (ethology) ,Peptide ,Microscopy, Atomic Force ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,Humans ,Molecular Biology ,Phospholipids ,chemistry.chemical_classification ,Calorimetry, Differential Scanning ,Chemistry ,Atomic force microscopy ,Circular Dichroism ,Milk Proteins ,Models, Structural ,Membrane ,Biochemistry ,Antigens, Surface ,Biophysics ,Chromatography, Gel - Abstract
Medin, a recently discovered 5.5 kDa peptide, is associated with amyloid deposits in the medial layer of human arteries and the prevalence is nearly 100% within individuals above 50 years. Presently, not much is known about its biochemical and biophysical properties or its pathway from soluble peptide to insoluble amyloid. Here we have characterized the behavior of medin in the presence of lipid membranes, using circular dichroism, isothermal titration calorimetry, differential scanning calorimetry, size exclusion chromatography, and atomic force microscopy (AFM). Medin was shown to exist as a monomer in solution with a predominantly random-coil structure. It binds lipid vesicles that have either a neutral or a negative surface potential. Upon association to membranes containing acidic lipids, it undergoes an electrostatically driven conformational change towards a mainly alpha-helical state. Prolonged incubation converts medin from an alpha-helical structure into an amyloid beta-sheet fibrillar state as confirmed by AFM. Based on these findings, we propose a mechanism of medin-amyloid formation where medin electrostatically associates in its monomeric form to biological interfaces displaying a negative potential. This process both increases the local peptide concentration and induces an aggregation-prone alpha-helical fold.
- Published
- 2007
45. Amide solvent protection analysis demonstrates that amyloid-beta(1-40) and amyloid-beta(1-42) form different fibrillar structures under identical conditions
- Author
-
Anders Öhman, Malin Lindhagen-Persson, Anders Olofsson, and A. Elisabeth Sauer-Eriksson
- Subjects
Magnetic Resonance Spectroscopy ,Amyloid β ,Stereochemistry ,Protein Conformation ,Microscopy, Atomic Force ,Biochemistry ,Residue (chemistry) ,chemistry.chemical_compound ,Alzheimer Disease ,Amide ,Humans ,Amino Acid Sequence ,Molecular Biology ,Amyloid beta-Peptides ,Atomic force microscopy ,Chemistry ,Neurofibrillary Tangles ,Cell Biology ,Amyloid β peptide ,Peptide Fragments ,Solvent ,Solutions ,Crystallography ,Structural biology ,Solvents ,Hydrogen–deuterium exchange ,Research Article - Abstract
AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39-43 residue long Abeta (amyloid-beta)-peptide. The most abundant species, Abeta(1-40) and Abeta(1-42), are both present within senile plaques, but Abeta(1-42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Abeta-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Abeta(1-40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Abeta(1-42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Abeta(1-40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two beta-strands connected with a bend. This protection pattern partly resembles the pattern found in Abeta(1-42) fibrils, but the Abeta(1-40) fibrils display a significantly increased protection for the N-terminal residues Phe4-His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37-Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.
- Published
- 2007
46. Signal sequence binding to the archaeal signal recognition particle
- Author
-
Tobias Hainzl and Elisabeth Sauer-Eriksson
- Subjects
Inorganic Chemistry ,Physics ,Signal recognition particle ,Structural Biology ,Signal sequence binding ,General Materials Science ,Physical and Theoretical Chemistry ,Condensed Matter Physics ,Biological system ,Biochemistry - Published
- 2015
47. Conformational variability of the GTPase domain of the signal recognition particle receptor FtsY
- Author
-
Talal Gariani, Tore Samuelsson, and A. Elisabeth Sauer-Eriksson
- Subjects
Adenosine Triphosphatases ,Models, Molecular ,Signal recognition particle ,GTP' ,Molecular Sequence Data ,Mycoplasma mycoides ,Receptors, Cytoplasmic and Nuclear ,Sequence alignment ,GTPase ,Biology ,Crystallography, X-Ray ,Transport protein ,GTP Phosphohydrolases ,Protein Structure, Tertiary ,Structure-Activity Relationship ,Biochemistry ,Bacterial Proteins ,Structural Biology ,Biophysics ,Amino Acid Sequence ,Receptor ,Signal recognition particle receptor ,Peptide sequence ,Sequence Alignment ,Signal Recognition Particle - Abstract
The prokaryotic signal recognition particle Ffh and its receptor FtsY allow targeting of proteins into or across the plasma membrane. The targeting process is GTP dependent and the two proteins constitute a distinct GTPase family. The receptor FtsY is composed of A and NG domains where the NG's GTPase domain plays a critical role in the targeting process. In this study, we describe two X-ray structures determined independently of each other of the NG domain of FtsY from Mycoplasma mycoides (MmFtsY). The two structures are markedly different in three of the nucleotide-binding segments, GI (P-loop), GII, and GIII, making only one of the structures compatible with nucleotide binding. Interestingly, the two distinct conformations of the nucleotide-binding segments of MmFtsY are similar to the apo- and ADP-loaded forms of certain ATPases. The structure of the extended interface between the A and NG domains of MmFtsY provides new insights into the role of the A domain for phospholipid interaction.
- Published
- 2005
48. The beta-strand D of transthyretin trapped in two discrete conformations
- Author
-
Erik Lundgren, Therese Eneqvist, Andreas Hörnberg, Anders Olofsson, and A. Elisabeth Sauer-Eriksson
- Subjects
Models, Molecular ,Protein Denaturation ,Amyloid ,Protein Conformation ,Biophysics ,Crystal structure ,Crystallography, X-Ray ,Biochemistry ,Analytical Chemistry ,Amyloid disease ,Protein structure ,Molecule ,Humans ,Prealbumin ,Molecular Biology ,biology ,Chemistry ,Hydrogen bond ,nutritional and metabolic diseases ,Space group ,Hydrogen Bonding ,Hydrogen-Ion Concentration ,Transthyretin ,Crystallography ,Mutation ,biology.protein ,Dimerization - Abstract
Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.
- Published
- 2004
49. Disulfide-bond formation in the transthyretin mutant Y114C prevents amyloid fibril formation in vivo and in vitro
- Author
-
Therese Eneqvist, Erik Lundgren, A. Elisabeth Sauer-Eriksson, Jana Jass, Yukio Ando, Taihei Miyakawa, Anders Olofsson, and Shoichi Katsuragi
- Subjects
Adult ,Amyloid ,Protein Conformation ,Mutant ,Crystallography, X-Ray ,Biochemistry ,chemistry.chemical_compound ,Epitopes ,Protein structure ,Humans ,Prealbumin ,Cysteine ,Disulfides ,Amyloid Neuropathies, Familial ,biology ,Wild type ,Antibodies, Monoclonal ,Middle Aged ,Recombinant Proteins ,Congo red ,Transthyretin ,Amyloid Neuropathy ,chemistry ,Amino Acid Substitution ,biology.protein ,Biophysics ,Mutagenesis, Site-Directed ,Tyrosine ,Electrophoresis, Polyacrylamide Gel ,Female ,Oxidation-Reduction ,Ex vivo - Abstract
The Y114C mutation in human transthyretin (TTR) is associated with a particular form of familial amyloidotic polyneuropathy. We show that vitreous aggregates ex vivo consist of either regular amyloid fibrils or disordered disulfide-linked precipitates that maintain the ability to bind Congo red. Furthermore, we demonstrate in vitro that the ATTR Y114C mutant exists in three forms: one unstable but nativelike tetrameric form, one highly aggregated form in which a network of disulfide bonds is formed, and one fibrillar form. The disulfide-linked aggregates and the fibrillar form of the mutant can be induced by heat induction under nonreduced and reduced conditions, respectively. Both forms are recognized by the amyloid specific antibody MAB(39-44). In a previous study, we have linked exposure of this epitope in TTR to a three-residue shift in beta-strand D. The X-ray crystallographic structure of reduced tetrameric ATTR Y114C shows a structure similar to that of the wild type but with a more buried position of Cys10 and with beta-mercaptoethanol associated with Cys114, verifying the strong tendency for this residue to form disulfide bonds. Combined with the ex vivo data, our in vitro findings suggest that ATTR Y114C can lead to disease either by forming regular unbranched amyloid fibrils or by forming disulfide-linked aggregates that maintain amyloid-like properties but are unable to form regular amyloid fibrils.
- Published
- 2002
50. A comparative analysis of 23 structures of the amyloidogenic protein transthyretin
- Author
-
Therese Eneqvist, Erik Lundgren, Anders Olofsson, A. Elisabeth Sauer-Eriksson, and Andreas Hörnberg
- Subjects
Models, Molecular ,endocrine system ,Amyloid ,Molecular Sequence Data ,Static Electricity ,Plaque, Amyloid ,Crystallography, X-Ray ,Protein Structure, Secondary ,Structural Biology ,medicine ,Humans ,Prealbumin ,Molecular Biology ,Protein secondary structure ,biology ,Chemistry ,Amyloidosis ,Point mutation ,nutritional and metabolic diseases ,Genetic Variation ,Water ,Hydrogen Bonding ,computer.file_format ,Hydrogen-Ion Concentration ,Protein Data Bank ,medicine.disease ,Protein tertiary structure ,Transport protein ,Protein Structure, Tertiary ,Transthyretin ,Biochemistry ,Solubility ,Mutation ,biology.protein ,Solvents ,computer ,Dimerization - Abstract
Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.
- Published
- 2000
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