| 41. |
- Holmner, Åsa, et al.
(författare)
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Blood group antigen recognition by Escherichia coli heat-labile enterotoxin
- 2007
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Ingår i: Journal of Molecular Biology. - London : Elsevier BV. - 0022-2836 .- 1089-8638. ; 371:3, s. 754-764
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Tidskriftsartikel (refereegranskat)abstract
- In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 angstrom crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAc alpha 3(Fuc alpha-2)Gal beta 4(Fuc alpha-3)Glc beta provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed. (c) 2007 Elsevier Ltd. All rights reserved.
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| 42. |
- Hoyer, Wolfgang, 1975, et al.
(författare)
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Interaction of Alzheimer's A beta peptide with an engineered binding protein--thermodynamics and kinetics of coupled folding-binding.
- 2008
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 378:2, s. 398-411
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Tidskriftsartikel (refereegranskat)abstract
- The oligomerization and aggregation of the amyloid-beta (A beta) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of A beta-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying A beta interactions is currently very limited. We have earlier determined the structure of A beta (1-40) in complex with the affibody protein Z(A beta 3), a selected binding protein based on a three-helix bundle scaffold (Z domain). Z(A beta 3) is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. Z(A beta 3) binds to the central and C-terminal part of A beta (residues 17-36), which adopts a beta-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the Z(A beta 3):A beta (1-40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free Z(A beta 3) is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial Z(A beta 3):A beta (1-40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both A beta and Z(A beta 3) fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and A beta is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate feature of biomolecular interactions involving the central and C-terminal part of A beta. Options for improvement of Z(A beta) binding proteins are discussed.
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| 43. |
- Hubner, Isaac A., et al.
(författare)
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Common motifs and topological effects in the protein folding transition state.
- 2006
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 359:4, s. 1075-1085
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Tidskriftsartikel (refereegranskat)abstract
- Through extensive experiment, simulation, and analysis of protein S6 (IRIS), we find that variations in nucleation and folding pathway between circular permutations are determined principally by the restraints of topology and specific nucleation, and affected by changes in chain entropy. Simulations also relate topological features to experimentally measured stabilities. Despite many sizable changes in Φ values and the structure of the transition state ensemble that result from permutation, we observe a common theme: the critical nucleus in each of the mutants share a subset of residues that can be mapped to the critical nucleus residues of the wild-type. Circular permutations create new N and C termini, which are the location of the largest disruption of the folding nucleus, leading to a decrease in both Φ values and the role in nucleation. Mutant nuclei are built around the wild-type nucleus but are biased towards different parts of the S6 structure depending on the topological and entropic changes induced by the location of the new N and C termini.
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| 44. |
- Högbom, Martin, et al.
(författare)
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Crystal Structure of Conserved Domains 1 and 2 of the Human DEAD-box Helicase DDX3X in Complex with the Mononucleotide AMP
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 372:1, s. 150-159
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Tidskriftsartikel (refereegranskat)abstract
- DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central β-sheet flanked by α-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPγS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPγS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.
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| 45. |
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| 46. |
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| 47. |
- Jeembaeva, Meerim, et al.
(författare)
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Osmotic pressure: resisting or promoting DNA ejection from phage?
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 381:2, s. 310-323
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Tidskriftsartikel (refereegranskat)abstract
- Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I in the course of ejection. In this work, we argue by a combination of experimental techniques (osmotic suppression without DNase I monitored by UV absorbance, pulse-field electrophoresis, and cryo-transmission electron microscopy visualization) and simple scaling modeling that intact genome (i.e., undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed.
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| 48. |
- Jenvert, Rose-Marie, et al.
(författare)
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The flexible n-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 365:3, s. 764-772
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Tidskriftsartikel (refereegranskat)abstract
- The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in 2 stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S (Delta L11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.
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| 49. |
- Jin, L H, et al.
(författare)
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Asymmetric allosteric activation of the symmetric ArgR hexamer
- 2005
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 346:1, s. 43-56
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Tidskriftsartikel (refereegranskat)abstract
- Hexameric arginine repressor, ArgR, bound to L-arginine serves both as the master transcriptional repressor/activator at diverse regulons in a wide range of bacteria and as a required cofactor for resolution of ColE1 plasmid multimers. Multifunctional ArgR is thus unusual in possessing features of specific gene regulators, global regulators, and non-specific gene organizers; its closest functional analog is probably CAP, the cyclic AMP receptor/activator protein. Isothermal titration calorimetry, surface plasmon resonance, and proteolysis indicate that binding of a single L-arginine residue per ArgR hexamer triggers a global conformational change and resets the affinities of the remaining five sites, making them 100-fold weaker. The analysis suggests a novel thermodynamic signature for this mechanism of activation. (C) 2004 Elsevier Ltd. All rights reserved.
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| 50. |
- Johansson, Tomas, et al.
(författare)
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X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli.
- 2005
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 352:2, s. 299-312
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Tidskriftsartikel (refereegranskat)abstract
- The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.
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