| 1. |
- Alm Rosenblad, Magnus, 1957, et al.
(författare)
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Detection of signal recognition particle (SRP) RNAs in the nuclear ribosomal internal transcribed spacer 1 (ITS1) of three lineages of ectomycorrhizal fungi (Agaricomycetes, Basidiomycota)
- 2016
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Ingår i: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; 13, s. 21-33
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Tidskriftsartikel (refereegranskat)abstract
- During a routine scan for Signal Recognition Particle (SRP) RNAs in eukaryotic sequences, we surprisingly found in silico evidence in GenBank for a 265-base long SRP RNA sequence in the ITS1 region of a total of 11 fully identified species in three ectomycorrhizal genera of the Basidiomycota (Fungi): Astraeus, Russula, and Lactarius. To rule out sequence artifacts, one specimen from a species indicated to have the SRP RNA-containing ITS region in each of these genera was ordered and re-sequenced. Sequences identical to the corresponding GenBank entries were recovered, or in the case of a non-original but conspecific specimen differed by three bases, showing that these species indeed have an SRP RNA sequence incorporated into their ITS1 region. Other than the ribosomal genes, this is the first known case of non-coding RNAs in the eukaryotic ITS region, and it may assist in the examination of other types of insertions in fungal genomes.
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| 2. |
- Aurelius, Oskar, et al.
(författare)
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The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.
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| 3. |
- Ding, Baojian, et al.
(författare)
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Sequence variation determining stereochemistry of a delta-11 desaturase active in moth sex pheromone biosynthesis
- 2016
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 1879-0240 .- 0965-1748. ; 74, s. 68-75
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Tidskriftsartikel (refereegranskat)abstract
- A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution.
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| 4. |
- Landreh, Michael, et al.
(författare)
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Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.
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| 5. |
- Costeira-Paulo, Joana, et al.
(författare)
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Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase
- 2018
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Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 25:3, s. 309-317
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Tidskriftsartikel (refereegranskat)abstract
- The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
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| 6. |
- Valegård, Karin, et al.
(författare)
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Structure of Rubisco from Arabidopsis thaliana in complex with 2-carboxyarabinitol-1,5-bisphosphate
- 2018
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Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 74:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Arabidopsis thaliana is reported at 1.5 Å resolution. In light of the importance of A. thaliana as a model organism for understanding higher plant biology, and the pivotal role of Rubisco in photosynthetic carbon assimilation, there has been a notable absence of an A. thaliana Rubisco crystal structure. A. thaliana Rubisco is an L8S8 hexadecamer comprising eight plastome-encoded catalytic large (L) subunits and eight nuclear-encoded small (S) subunits. A. thaliana produces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B), and this crystal structure provides a snapshot of A. thaliana Rubisco containing the low-abundance RbcS3B small-subunit isoform. Crystals were obtained in the presence of the transition-state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate. A. thaliana Rubisco shares the overall fold characteristic of higher plant Rubiscos, but exhibits an interesting disparity between sequence and structural relatedness to other Rubisco isoforms. These results provide the structural framework to understand A. thaliana Rubisco and the potential catalytic differences that could be conferred by alternative A. thaliana Rubisco small-subunit isoforms.
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| 7. |
- Hemsworth, Glyn R., et al.
(författare)
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Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut
- 2016
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Ingår i: Open Biology. - : Royal Society of London. - 2046-2441. ; 6:7
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Tidskriftsartikel (refereegranskat)abstract
- The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta) genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an alpha-xylosidase, a beta-glucosidase, and two alpha-L-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.
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| 8. |
- Larsbrink, Johan, 1982, et al.
(författare)
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A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin
- 2016
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:260
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Tidskriftsartikel (refereegranskat)abstract
- Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature’s most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown.ResultsHere we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor–regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm.ConclusionsBy combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.
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| 9. |
- Pilstål, Robert, 1985-
(författare)
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On protein structure, function and modularity from an evolutionary perspective
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We are compounded entities, given life by a complex molecular machinery. When studying these molecules we have to make sense of a diverse set of dynamical nanostructures with wast and intricate patterns of interactions. Protein polymers is one of the major groups of building blocks of such nanostructures which fold up into more or less distinct three dimensional structures. Due to their shape, dynamics and chemical properties proteins are able to perform a plethora of specific functions essential to all known cellular lifeforms.The connection between protein sequence, translated into protein structure and in the continuation into protein function is well accepted but poorly understood. Malfunction in the process of protein folding is known to be implicated in natural aging, cancer and degenerative diseases such as Alzheimer's.Protein folds are described hierarchically by structural ontologies such as SCOP, CATH and Pfam all which has yet to succeed in deciphering the natural language of protein function. These paradigmatic views centered on protein structure fail to describe more mutable entities, such as intrinsically disordered proteins (IDPs) which lack a clear defined structure.As of 2012, about two thirds of cancer patients was predicted to survive past 5 years of diagnosis. Despite this, about a third do not survive and numerous of successfully treated patients suffer from secondary conditions due to chemotherapy, surgery and the like. In order to handle cancer more efficiently we have to better understand the underlying molecular mechanisms.Elusive to standard methods of investigation, IDPs have a central role in pathology; dysfunction in IDPs are key factors in cellular system failures such as cancer, as many IDPs are hub regulators for major cell functions. These IDPs carry short conserved functional boxes, that are not described by known ontologies, which suggests the existence of a smaller entity. In an investigation of a pair of such boxes of c-MYC, a plausible structural model of its interacting with Pin1 emerged, but such a model still leaves the observer with a puzzle of understanding the actual function of that interaction.If the protein is represented as a graph and modeled as the interaction patterns instead of as a structural entity, another picture emerges. As a graph, there is a parable from that of the boxes of IDPs, to that of sectors of allosterically connected residues and the theory of foldons and folding units. Such a description is also useful in deciphering the implications of specific mutations.In order to render a functional description feasible for both structured and disordered proteins, there is a need of a model separate from form and structure. Realized as protein primes, patterns of interaction, which has a specific function that can be defined as prime interactions and context. With function defined as interactions, it might be possible that the discussion of proteins and their mechanisms is thereby simplified to the point rendering protein structural determination merely supplementary to understanding protein function.
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| 10. |
- Wu, Min, 1986, et al.
(författare)
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Proline 411 biases the conformation of the intrinsically disordered plant UVR8 photoreceptor C27 domain altering the functional properties of the peptide
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- UVR8 (UV RESISTANCE LOCUS 8) is a UV-B photoreceptor responsible for initiating UV-B signalling in plants. UVR8 is a homodimer in its signalling inactive form. Upon absorption of UV radiation, the protein monomerizes into its photoactivated state. In the monomeric form, UVR8 binds the E3 ubiquitin ligase COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1), triggering subsequent UV-B-dependent photomorphogenic development in plants. Recent in vivo experiments have shown that the UVR8 C-terminal region (aa 397-423; UVR8(C27)) alone is sufficient to regulate the activity of COP1. In this work, CD spectroscopy and NMR experiments showed that the UVR8(C27) domain was non-structured but gained secondary structure at higher temperatures leading to increased order. Bias-exchange metadynamics simulations were also performed to evaluate the free energy landscape of UVR8(C27). An inverted free energy landscape was revealed, with a disordered structure in the global energy minimum. Flanking the global energy minimum, more structured states were found at higher energies. Furthermore, stabilization of the low energy disordered state was attributed to a proline residue, P411, as evident from P411A mutant data. P411 is also a key residue in UVR8 binding to COP1. UVR8(C27) is therefore structurally competent to function as a molecular switch for interaction of UVR8 with different binding partners since at higher free energies different structural conformations are being induced in this peptide. P411 has a key role for this function.
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