| 1. |
- Edwin, Aaron, et al.
(författare)
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Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae
- 2015
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 24:12, s. 2076-2080
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Tidskriftsartikel (refereegranskat)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 alpha-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.
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| 2. |
- Forsgren, Nina, 1979-, et al.
(författare)
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Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB
- 2009
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 18:9, s. 1896-1905
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Tidskriftsartikel (refereegranskat)abstract
- The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V-) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V-domain from S. gordonii SspB at 2.3 A resolution. The domain comprises a beta-supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V-domain is similar to the previously reported V-domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V-domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V-domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.
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| 3. |
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| 4. |
- Kjaergaard, Magnus, et al.
(författare)
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Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris.
- 2007
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 16:9
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Tidskriftsartikel (refereegranskat)abstract
- The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.
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| 5. |
- Leppert, Axel, et al.
(författare)
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ATP-independent molecular chaperone activity generated under reducing conditions
- 2022
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Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:8
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Tidskriftsartikel (refereegranskat)abstract
- Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra- to intermolecular disulfide bond relay mechanism. Chaperone-active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol-containing compounds and proteins, and appear in parallel with reduction-induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space.Significance: Chaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction-induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.
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| 6. |
- Li, Ping, et al.
(författare)
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PcoB is a defense outer membrane protein that facilitates cellular uptake of copper
- 2022
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 31:7
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Tidskriftsartikel (refereegranskat)abstract
- Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.
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| 7. |
- Llinas, Paola, et al.
(författare)
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Structural studies of human alkaline phosphatase in complex with strontium : implication for its secondary effect in bones.
- 2006
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 15:7, s. 1691-700
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Tidskriftsartikel (refereegranskat)abstract
- Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.
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| 8. |
- Maxwell, Karen L., et al.
(författare)
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Protein folding : Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins
- 2005
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 14, s. 602-16
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Tidskriftsartikel (refereegranskat)abstract
- Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25°C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.
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| 9. |
- Otzen, Daniel E, et al.
(författare)
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Transient formation of nano-crystalline structures during fibrillation of an A-like peptide
- 2004
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 13, s. 1417-21
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Tidskriftsartikel (refereegranskat)abstract
- During the first few minutes of fibrillation of a 14-residue peptide homologous to the hydrophobic C-terminal part of the A-peptide, EM micrographs reveal small crystalline areas (100 to 150 nm, repeating unit 47 Å) scattered in more amorphous material. On a longer time scale, these crystalline areas disappear and are replaced by tangled clusters resembling protofilaments (hours), and eventually by more regular amyloid fibrils of 60 Å to 120 Å diameter (days). The transient population of the crystalline areas indicates the presence of ordered substructures in the early fibrillation process, the diameter of which matches the length of the 14-mer peptide in an extended -strand conformation.
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| 10. |
- Öhman, Anders, et al.
(författare)
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Solution structures and backbone dynamics of the ribosomal protein S6 and its permutant P54-55
- 2010
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 19:1, s. 183-189
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Tidskriftsartikel (refereegranskat)abstract
- The ribosomal protein S6 from Thermus thermophilus has served as a model system for the study of protein folding, especially for understanding the effects of circular permutations of secondary structure elements. This study presents the structure of a permutant protein, the 96-residue P54-55, and the structure of its 101-residue parent protein S6wt in solution. The data also characterizes the effects of circular permutation on the backbone dynamics of S6. Consistent with crystallographic data on S6wt, the overall solution structures of both P54-55 and S6wt show a β-sheet of four antiparallel β-strands with two α-helices packed on one side of the sheet. In clear contrast to the crystal data, however, the solution structure of S6wt reveals a disordered loop in the region between β-strands 2 and 3 (Leu43-Phe60) instead of a well-ordered stretch and associated hydrophobic mini-core observed in the crystal structure. Moreover, the data for P54-55 show that the joined wild-type N- and C-terminals form a dynamically robust stretch with a hairpin structure that complies with the in silico design. Taken together, the results explain why the loop region of the S6wt structure is relatively insensitive to mutational perturbations, and why P54-55 is more stable than S6wt: the permutant incision at Lys54-Asp55 is energetically neutral by being located in an already disordered loop whereas the new hairpin between the wild-type N- and C-termini is stabilizing.
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