Publications by authors named "Hans Hebert"

69 Publications

A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity.

J Mol Biol 2021 Jun 21;433(18):167114. Epub 2021 Jun 21.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), KAIST Institute of BioCentury, Daejeon 34141, Korea. Electronic address:

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.
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http://dx.doi.org/10.1016/j.jmb.2021.167114DOI Listing
June 2021

Publisher Correction: Crocus-derived compounds alter the aggregation pathway of Alzheimer's Disease - associated beta amyloid protein.

Sci Rep 2021 Feb 2;11(1):3315. Epub 2021 Feb 2.

GAIA Research Center, Bioanalytical Department, The Goulandris Natural History Museum, 14562, Kifissia, Greece.

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http://dx.doi.org/10.1038/s41598-021-82907-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854578PMC
February 2021

Cryo-EM structure of native human uromodulin, a zona pellucida module polymer.

EMBO J 2020 12 16;39(24):e106807. Epub 2020 Nov 16.

Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) "domain". Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo-electron microscopy study of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of heteromeric vertebrate egg coat filaments identify a common sperm-binding region at the interface between subunits.
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http://dx.doi.org/10.15252/embj.2020106807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737619PMC
December 2020

Crocus-derived compounds alter the aggregation pathway of Alzheimer's Disease: associated beta amyloid protein.

Sci Rep 2020 10 23;10(1):18150. Epub 2020 Oct 23.

GAIA Research Center, Bioanalytical Department, The Goulandris Natural History Museum, 14562, Kifissia, Greece.

Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)-based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aβ fibrillogenesis, a neuropathological hallmark of Alzheimer's Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aβ concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aβ aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aβ amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.
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http://dx.doi.org/10.1038/s41598-020-74770-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585429PMC
October 2020

The Polyglutamine Expansion at the N-Terminal of Huntingtin Protein Modulates the Dynamic Configuration and Phosphorylation of the C-Terminal HEAT Domain.

Structure 2020 09 14;28(9):1035-1050.e8. Epub 2020 Jul 14.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), KI for the BioCentury, Daejeon 34141, Korea. Electronic address:

The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural information on its HEAT-repeat domains. Here, we present analyses of the impact of polyQ length on the structure and function of HTT via an integrative structural and biochemical approach. The cryo-EM analysis of normal (Q23) and disease (Q78) type HTTs shows that the structures of apo HTTs significantly differ from the structure of HTT in a HAP40 complex and that the polyQ expansion induces global structural changes in the relative movements among the HTT domains. In addition, we show that the polyQ expansion alters the phosphorylation pattern across HTT and that Ser2116 phosphorylation in turn affects the global structure and function of HTT. These results provide a molecular basis for the effect of the polyQ segment on HTT structure and activity, which may be important for HTT pathology.
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http://dx.doi.org/10.1016/j.str.2020.06.008DOI Listing
September 2020

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions.

PLoS One 2020 9;15(7):e0228607. Epub 2020 Jul 9.

Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228607PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347166PMC
September 2020

Recombinant Bri3 BRICHOS domain is a molecular chaperone with effect against amyloid formation and non-fibrillar protein aggregation.

Sci Rep 2020 06 17;10(1):9817. Epub 2020 Jun 17.

Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Karolinska Institutet, Huddinge, Sweden.

Molecular chaperones assist proteins in achieving a functional structure and prevent them from misfolding into aggregates, including disease-associated deposits. The BRICHOS domain from familial dementia associated protein Bri2 (or ITM2B) probably chaperones its specific proprotein region with high β-sheet propensity during biosynthesis. Recently, Bri2 BRICHOS activity was found to extend to other amyloidogenic, fibril forming peptides, in particular, Alzheimer's disease associated amyloid-β peptide, as well as to amorphous aggregate forming proteins. However, the biological functions of the central nervous system specific homologue Bri3 BRICHOS are still to be elucidated. Here we give a detailed characterisation of the recombinant human (rh) Bri3 BRICHOS domain and compare its structural and functional properties with rh Bri2 BRICHOS. The results show that rh Bri3 BRICHOS forms more and larger oligomers, somewhat more efficiently prevents non-fibrillar protein aggregation, and less efficiently reduces Aβ42 fibril formation compared to rh Bri2 BRICHOS. This suggests that Bri2 and Bri3 BRICHOS have overlapping molecular mechanisms and that their apparently different tissue expression and processing may result in different physiological functions.
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http://dx.doi.org/10.1038/s41598-020-66718-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299998PMC
June 2020

Augmentation of Bri2 molecular chaperone activity against amyloid-β reduces neurotoxicity in mouse hippocampus in vitro.

Commun Biol 2020 01 20;3(1):32. Epub 2020 Jan 20.

Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Division of Neurogeriatrics, Karolinska Institutet, 141 57, Huddinge, Sweden.

Molecular chaperones play important roles in preventing protein misfolding and its potentially harmful consequences. Deterioration of molecular chaperone systems upon ageing are thought to underlie age-related neurodegenerative diseases, and augmenting their activities could have therapeutic potential. The dementia relevant domain BRICHOS from the Bri2 protein shows qualitatively different chaperone activities depending on quaternary structure, and assembly of monomers into high-molecular weight oligomers reduces the ability to prevent neurotoxicity induced by the Alzheimer-associated amyloid-β peptide 1-42 (Aβ42). Here we design a Bri2 BRICHOS mutant (R221E) that forms stable monomers and selectively blocks a main source of toxic species during Aβ42 aggregation. Wild type Bri2 BRICHOS oligomers are partly disassembled into monomers in the presence of the R221E mutant, which leads to potentiated ability to prevent Aβ42 toxicity to neuronal network activity. These results suggest that the activity of endogenous molecular chaperones may be modulated to enhance anti-Aβ42 neurotoxic effects.
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http://dx.doi.org/10.1038/s42003-020-0757-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971075PMC
January 2020

Aldehyde-alcohol dehydrogenase forms a high-order spirosome architecture critical for its activity.

Nat Commun 2019 10 4;10(1):4527. Epub 2019 Oct 4.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Korea.

Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity.
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http://dx.doi.org/10.1038/s41467-019-12427-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778083PMC
October 2019

CryoEM: a crystals to single particles round-trip.

Authors:
Hans Hebert

Curr Opin Struct Biol 2019 10 21;58:59-67. Epub 2019 Jun 21.

School of Engineering Sciences in Chemistry, Biotechnology and Health, Department of Biomedical Engineering and Health Systems, KTH Royal Institute of Technology, Sweden; Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 11, S-141 52 Huddinge, Sweden. Electronic address:

In the era of intense and steadily increasing attention to cryo electron microscopy (cryoEM) as a powerful tool in structural biology, particularly with regard to randomly oriented biological macromolecules, studies of 2D and small 3D crystals using cryoEM provide added value for addressing-specific questions. Size and shape demands are not as restrictive as for single particle specimens. Crystallization may stabilize whole or partly flexible molecules. Resolutions beyond 2Å, for 3D crystals even sub-Ångström structures, can be obtained allowing studies of chemical properties in detail. The electron dose can be kept low and reduce radiation damage for sensitive specimens. In contrast to X-ray crystallography, scattering of electrons will be directly related to the Coulomb potential and thus give information about charge distribution in biomolecules.
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http://dx.doi.org/10.1016/j.sbi.2019.05.008DOI Listing
October 2019

Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase.

Genes Dev 2019 06 28;33(11-12):620-625. Epub 2019 Mar 28.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea.

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.
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http://dx.doi.org/10.1101/gad.323790.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546062PMC
June 2019

A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein.

FEBS J 2018 05 14;285(10):1873-1885. Epub 2018 Apr 14.

Division for Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Huddinge, Sweden.

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide β17. The fusion protein NT*-β17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that β17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopts a β-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.
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http://dx.doi.org/10.1111/febs.14451DOI Listing
May 2018

Structural modelling of the DNAJB6 oligomeric chaperone shows a peptide-binding cleft lined with conserved S/T-residues at the dimer interface.

Sci Rep 2018 03 26;8(1):5199. Epub 2018 Mar 26.

Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, PO Box 124, SE-221 00, Lund, Sweden.

The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface. Mixed isotope crosslinking showed that the oligomers are dynamic entities that exchange subunits. The purified protein is well folded, soluble and composed of oligomers with a varying number of subunits according to small-angle X-ray scattering (SAXS). Elongated particles (160 × 120 Å) were detected by electron microscopy and single particle reconstruction resulted in a density map of 20 Å resolution into which the DNAJB6 dimers fit. The structure of the oligomer and the S/T-rich region is of great importance for the understanding of the function of DNAJB6 and how it can bind aggregation-prone peptides and prevent amyloid diseases.
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http://dx.doi.org/10.1038/s41598-018-23035-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979959PMC
March 2018

Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers.

J Biol Chem 2018 04 13;293(15):5746-5754. Epub 2018 Feb 13.

From the Department of Medical Biochemistry, University of Gothenburg, 40530 Gothenburg, Sweden,

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.
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http://dx.doi.org/10.1074/jbc.RA117.001014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900763PMC
April 2018

Integrative Structural Investigation on the Architecture of Human Importin4_Histone H3/H4_Asf1a Complex and Its Histone H3 Tail Binding.

J Mol Biol 2018 03 31;430(6):822-841. Epub 2018 Jan 31.

Department of Biological Sciences, Cancer Metastasis Control Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea. Electronic address:

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The cross-linking mass spectrometry data showed that the C-terminal region of Importin4 was extensively cross-linked with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic patch in Importin4 accommodates the histone H3 tail, and that histone H3 Lys14 contributes to the interaction with Importin4. In addition, we show that Asf1a modulates the binding of histone H3/H4 to Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.
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http://dx.doi.org/10.1016/j.jmb.2018.01.015DOI Listing
March 2018

Bri2 BRICHOS client specificity and chaperone activity are governed by assembly state.

Nat Commun 2017 12 12;8(1):2081. Epub 2017 Dec 12.

Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Division of Neurogeriatrics, Karolinska Institutet, 141 57, Huddinge, Sweden.

Protein misfolding and aggregation is increasingly being recognized as a cause of disease. In Alzheimer's disease the amyloid-β peptide (Aβ) misfolds into neurotoxic oligomers and assembles into amyloid fibrils. The Bri2 protein associated with Familial British and Danish dementias contains a BRICHOS domain, which reduces Aβ fibrillization as well as neurotoxicity in vitro and in a Drosophila model, but also rescues proteins from irreversible non-fibrillar aggregation. How these different activities are mediated is not known. Here we show that Bri2 BRICHOS monomers potently prevent neuronal network toxicity of Aβ, while dimers strongly suppress Aβ fibril formation. The dimers assemble into high-molecular-weight oligomers with an apparent two-fold symmetry, which are efficient inhibitors of non-fibrillar protein aggregation. These results indicate that Bri2 BRICHOS affects qualitatively different aspects of protein misfolding and toxicity via different quaternary structures, suggesting a means to generate molecular chaperone diversity.
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http://dx.doi.org/10.1038/s41467-017-02056-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727130PMC
December 2017

The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin.

Biochem Biophys Res Commun 2017 10 30;492(3):331-337. Epub 2017 Aug 30.

Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden. Electronic address:

To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles.
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http://dx.doi.org/10.1016/j.bbrc.2017.08.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596833PMC
October 2017

Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation.

Sci Rep 2017 08 11;7(1):7897. Epub 2017 Aug 11.

Department of Biosciences and Nutrition, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, SE-141 83, Huddinge, Sweden.

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.
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http://dx.doi.org/10.1038/s41598-017-07912-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554250PMC
August 2017

Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations.

Biochim Biophys Acta Gen Subj 2017 Nov 25;1861(11 Pt A):2726-2738. Epub 2017 Jul 25.

Department of Experimental Medical Science, Lund University, Lund, Sweden. Electronic address:

Background: Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) is a key protein for the transport of cholesterol from the vascular wall to the liver. The formation and structure of nascent HDL, composed of apoA-I and phospholipids, is critical to this process.

Methods: The HDL was assembled in vitro from apoA-I, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a 1:4:50 molar ratio. The structure of HDL was investigated in vitreous samples, frozen at cryogenic temperatures, as well as in negatively stained samples by transmission electron microscopy. Low resolution electron density maps were next used as restraints in biased Monte Carlo simulations of apolipoprotein A-I dimers, with an initial structure derived from atomic resolution X-ray structures.

Results: Two final apoA-I structure models for the full-length structure of apoA-I dimer in the lipid bound conformation were generated, showing a nearly circular, flat particle with an uneven particle thickness.

Conclusions: The generated structures provide evidence for the discoidal, antiparallel arrangement of apoA-I in nascent HDL, and propose two preferred conformations of the flexible N-termini.

General Significance: The novel full-length structures of apoA-I dimers deepens the understanding to the structure-function relationship of nascent HDL with significance for the prevention of lipoprotein-related disease. The biased simulation method used in this study provides a powerful and convenient modelling tool with applicability for structural studies and modelling of other proteins and protein complexes.
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http://dx.doi.org/10.1016/j.bbagen.2017.07.017DOI Listing
November 2017

Efficient protein production inspired by how spiders make silk.

Nat Commun 2017 05 23;8:15504. Epub 2017 May 23.

Division for Neurogeriatrics, Department of NVS, Center for Alzheimer Research, Karolinska Institutet, 141 57 Huddinge, Sweden.

Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
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http://dx.doi.org/10.1038/ncomms15504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457526PMC
May 2017

RNA activation-independent DNA targeting of the Type III CRISPR-Cas system by a Csm complex.

EMBO Rep 2017 05 31;18(5):826-840. Epub 2017 Mar 31.

Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea

The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from is reconstituted with a minimalistic combination of Csm12345, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.
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http://dx.doi.org/10.15252/embr.201643700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412804PMC
May 2017

Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity.

J Biol Chem 2017 05 21;292(19):8103-8121. Epub 2017 Mar 21.

From the Departments of Biochemistry and Structural Biology and

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IVI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IVI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.
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http://dx.doi.org/10.1074/jbc.M116.766816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427286PMC
May 2017

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy.

J Vis Exp 2017 03 5(121). Epub 2017 Mar 5.

Department of Biosciences and Nutrition, Karolinska Institutet; School of Technology and Health, KTH Royal Institute of Technology;

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca-induced binding of 5-lipoxygenase on nanodiscs is presented.
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http://dx.doi.org/10.3791/55148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409284PMC
March 2017

Biomimetic spinning of artificial spider silk from a chimeric minispidroin.

Nat Chem Biol 2017 03 9;13(3):262-264. Epub 2017 Jan 9.

Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.

Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.
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http://dx.doi.org/10.1038/nchembio.2269DOI Listing
March 2017

Molecular Architecture of  Yeast Chromatin Assembly Factor 1.

Sci Rep 2016 05 25;6:26702. Epub 2016 May 25.

Department of Biological Sciences, KAIST Institute for the BioCentury, Cancer Metastasis Control Center, KAIST, 291 Daehakro Yuseong Daejeon, 34141, Korea.

Chromatin Assembly Complex 1 (CAF-1) is a major histone chaperone involved in deposition of histone H3 and H4 into nucleosome. CAF-1 is composed of three subunits; p150, p60 and p48 for human and Cac1, Cac2 and Cac3 for yeast. Despite of its central role in chromatin formation, structural features of the full CAF-1 in complex with histones and other chaperones have not been well characterized. Here, we dissect molecular architecture of yeast CAF-1 (yCAF-1) by cross-linking mass spectrometry (XL-MS) and negative stain single-particle electron microscopy (EM). Our work revealed that Cac1, the largest subunit of yCAF-1, might serve as a major histone binding platform linking Cac2 and Cac3. In addition, EM analysis showed that yCAF-1 adopts a bilobal shape and Cac1 connecting Cac2 and Cac3 to generate a platform for binding histones. This study provides the first structural glimpse of the full CAF-1 complex and a structural framework to understand histone chaperoning processes.
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http://dx.doi.org/10.1038/srep26702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879628PMC
May 2016

Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy.

Micron 2016 Jun 1;85:39-43. Epub 2016 Apr 1.

School of Technology and Health, KTH Royal Institute of Technology and Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden. Electronic address:

Intravenously injected microbubbles (MBs) can be utilized as ultrasound contrast agent (CA) resulting in enhanced image quality. A novel CA, consisting of air filled MBs stabilized with a shell of polyvinyl alcohol (PVA) has been developed. These spherical MBs have been decorated with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as both ultrasound and magnetic resonance imaging (MRI) CA. In this study, a mathematical model was introduced that determined the shell thickness of two types of SPIONs decorated MBs (Type A and Type B). The shell thickness of MBs is important to determine, as it affects the acoustical properties. In order to investigate the shell thickness, thin sections of plastic embedded MBs were prepared and imaged using transmission electron microscopy (TEM). However, the sections were cut at random distances from the MB center, which affected the observed shell thickness. Hence, the model determined the average shell thickness of the MBs from corrected mean values of the outer and inner radii observed in the TEM sections. The model was validated using simulated slices of MBs with known shell thickness and radius. The average shell thickness of Type A and Type B MBs were 651nm and 637nm, respectively.
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http://dx.doi.org/10.1016/j.micron.2016.03.009DOI Listing
June 2016

Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs.

PLoS One 2016 24;11(3):e0152116. Epub 2016 Mar 24.

Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden.

An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron microscopy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152116PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806843PMC
August 2016

Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function.

Elife 2016 Mar 22;5:e11184. Epub 2016 Mar 22.

Center for Human Genetic Research, Massachusetts General Hospital, Boston, United States.

The polyglutamine expansion in huntingtin protein causes Huntington's disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single-particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical α-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases α-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.
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http://dx.doi.org/10.7554/eLife.11184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846397PMC
March 2016

Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation.

Biomed Res Int 2015 27;2015:693869. Epub 2015 Aug 27.

Department of Biosciences and Nutrition, Karolinska Institutet, Novum, 14183 Huddinge, Sweden ; School of Technology and Health, KTH Royal Institute of Technology, Novum, 14183 Huddinge, Sweden.

Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered two-dimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection.
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http://dx.doi.org/10.1155/2015/693869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564634PMC
July 2016

Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

Colloids Surf B Biointerfaces 2015 Nov 17;135:90-98. Epub 2015 Jul 17.

CEB - Centre of Biological Engineering, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal. Electronic address:

Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.
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http://dx.doi.org/10.1016/j.colsurfb.2015.06.073DOI Listing
November 2015