Publications by authors named "Pietro Roversi"

91 Publications

Cooperative stabilisation of 14-3-3σ protein-protein interactions covalent protein modification.

Chem Sci 2021 Oct 6;12(39):12985-12992. Epub 2021 Sep 6.

Leicester Institute for Structural and Chemical Biology, University of Leicester University Road Leicester LE1 7RH UK

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes a large network of interactions with partner proteins. Many of these protein-protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like 'molecular glues' is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth.
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http://dx.doi.org/10.1039/d1sc02120fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8513901PMC
October 2021

Anharmonic Thermal Motion Modelling in the Experimental XRD Charge Density Determination of 1-Methyluracil at T = 23 K.

Molecules 2021 May 21;26(11). Epub 2021 May 21.

Chemistry Department, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.

The experimental electron density distribution (EDD) of 1-methyluracil (1-MUR) was obtained by single crystal X-ray diffraction (XRD) experiments at 23 K. Four different structural models fitting an extensive set of XRD data to a resolution of (sinθ/λ) = 1.143 Å are compared. Two of the models include anharmonic temperature factors, whose inclusion is supported by the Hamilton test at a 99.95% level of confidence. Positive Fourier residuals up to 0.5 eÅ in magnitude were found close to the methyl group and in the region of hydrogen bonds. Residual density analysis (RDA) and molecular dynamics simulations in the solid-state demonstrate that these residuals can be likely attributed to unresolved disorder, possibly dynamical and long-range in nature. Atomic volumes and charges, molecular moments up to hexadecapoles, as well as maps of the molecular electrostatic potential were obtained from distributed multipole analysis of the EDD. The derived electrostatic properties neither depend on the details of the multipole model, nor are significantly affected by the explicit inclusion of anharmonicity in the least-squares model. The distribution of atomic charges in 1-MUR is not affected by the crystal environment in a significant way. The quality of experimental findings is discussed in light of in-crystal and gas-phase quantum simulations.
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http://dx.doi.org/10.3390/molecules26113075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196607PMC
May 2021

Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase.

Structure 2021 04 21;29(4):357-370.e9. Epub 2020 Dec 21.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester, LE1 7RH,, UK. Electronic address:

UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70-80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.
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http://dx.doi.org/10.1016/j.str.2020.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024514PMC
April 2021

Partial catalytic Cys oxidation of human GAPDH to Cys-sulfonic acid.

Wellcome Open Res 2020 25;5:114. Epub 2020 Aug 25.

Leicester Institute of Chemical and Structural Biology and Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, LE1 7HB, UK.

: n-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the NAD -dependent oxidative phosphorylation of n-glyceraldehyde-3-phosphate to 1,3-diphospho-n-glycerate and its reverse reaction in glycolysis and gluconeogenesis. : Four distinct crystal structures of human n-Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) have been determined from protein purified from the supernatant of HEK293F human epithelial kidney cells. : X-ray crystallography and mass-spectrometry indicate that the catalytic cysteine of the protein ( GAPDH Cys152) is partially oxidised to cysteine S-sulfonic acid. The average occupancy for the Cys152-S-sulfonic acid modification over the 20 crystallographically independent copies of GAPDH across three of the crystal forms obtained is 0.31±0.17. : The modification induces no significant structural changes on the tetrameric enzyme, and only makes aspecific contacts to surface residues in the active site, in keeping with the hypothesis that the oxidising conditions of the secreted mammalian cell expression system result in GAPDH catalytic cysteine S-sulfonic acid modification and irreversible inactivation of the enzyme.
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http://dx.doi.org/10.12688/wellcomeopenres.15893.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422855PMC
August 2020

Erratum to "Modulation of ERQC and ERAD: A Broad-Spectrum Spanner in the Works of Cancer Cells?"

J Oncol 2020 9;2020:1396429. Epub 2020 Jul 9.

Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 7RH, UK.

[This corrects the article DOI: 10.1155/2019/8384913.].
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http://dx.doi.org/10.1155/2020/1396429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368190PMC
July 2020

Targeting Endoplasmic Reticulum α-Glucosidase I with a Single-Dose Iminosugar Treatment Protects against Lethal Influenza and Dengue Virus Infections.

J Med Chem 2020 04 15;63(8):4205-4214. Epub 2020 Apr 15.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England, U.K.

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of -(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00067DOI Listing
April 2020

Modulation of ERQC and ERAD: A Broad-Spectrum Spanner in the Works of Cancer Cells?

J Oncol 2019 1;2019:8384913. Epub 2019 Oct 1.

Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 7RH, UK.

Endoplasmic reticulum glycoprotein folding quality control (ERQC) and ER-associated degradation (ERAD) preside over cellular glycoprotein secretion and maintain steady glycoproteostasis. When cells turn malignant, cancer cell plasticity is affected and supported either by point mutations, preferential isoform selection, altered expression levels, or shifts to conformational equilibria of a secreted glycoprotein. Such changes are crucial in mediating altered extracellular signalling, metabolic behavior, and adhesion properties of cancer cells. It is therefore conceivable that interference with ERQC and/or ERAD can be used to selectively damage cancers. Indeed, inhibitors of the late stages of ERAD are already in the clinic against cancers such as multiple myeloma. Here, we review recent advances in our understanding of the complex relationship between glycoproteostasis and cancer biology and discuss the potential of ERQC and ERAD modulators for the selective targeting of cancer cell plasticity.
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http://dx.doi.org/10.1155/2019/8384913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791201PMC
October 2019

Early management of COPD: where are we now and where do we go from here? A Delphi consensus project.

Int J Chron Obstruct Pulmon Dis 2019;14:353-360. Epub 2019 Feb 4.

Istituti Clinici Scientifici Maugeri, IRCCS di Cassano delle Murge, Cassano delle Murge (BA), Italy.

Purpose: There is a lack of consensus on the most appropriate early diagnostic strategy, criteria for early access to treatment and follow-up approach for patients with COPD.

Materials And Methods: A Delphi consensus project investigated the early management of COPD. We formulated two questionnaires for completion by pneumologists in Italy.

Results: A total of 207 specialists completed questionnaire 1 and 184 of them questionnaire 2, between November 2016 and October 2017. Early diagnosis of COPD was considered uncommon for 93.2% of the expert panel. Regardless of the definition of "early diagnosis" - a diagnosis made before the clinical manifestation of the disease for most responders (60.4%) - experts were confident of the positive effects of early disease management, which they consider is effective in modifying the natural history of the disease. Lack of awareness of the disease was considered the first limiting factor to early COPD management for 78% of respondents. The most effective steps to reduce functional decline were considered to be smoking cessation, followed by long-acting β2-agonist (LABA)/long-acting muscarinic antagonist (LAMA), LAMA, LABA, and finally inhaled corticosteroid/LABA (<0.01 for each paired comparison). Specialists considered it "inappropriate" for general practitioners to perform both the early diagnosis and therapy of COPD without the involvement of a specialist.

Conclusion: Early management of COPD is uncommon, and although data on the effects of early disease management on long-term outcomes are limited, Italian experts are confident of the clinical efficacy of this approach.
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http://dx.doi.org/10.2147/COPD.S176662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366359PMC
July 2019

EFR-Mediated Innate Immune Response in is a Useful Tool for Identification of Novel ERQC Modulators.

Genes (Basel) 2018 Dec 27;10(1). Epub 2018 Dec 27.

Institute of Sciences of Food Production, C.N.R. Unit of Lecce, via Monteroni, I-73100 Lecce, Italy.

Plants offer a simpler and cheaper alternative to mammalian animal models for the study of endoplasmic reticulum glycoprotein folding quality control (ERQC). In particular, the () innate immune response to bacterial peptides provides an easy means of assaying ERQC function in vivo. A number of mutants that are useful to study ERQC have been described in the literature, but only for a subset of these mutants the innate immune response to bacterial elicitors has been measured beyond monitoring plant weight and some physio-pathological parameters related to the plant immune response. In order to probe deeper into the role of ERQC in the plant immune response, we monitored expression levels of the Phosphate-induced 1 ( and reticulin-oxidase homologue ( genes in the ER α-Glu II and the UGGT mutant plants, in response to bacterial peptides elf18 and flg22. The elf18 response was impaired in the but not completely abrogated in the mutant plants, raising the possibility that the latter enzyme is partly dispensable for EF-Tu receptor (EFR) signaling. In the mutant, seedling growth was impaired only by concomitant application of the ER α-Glu II B-DNJ inhibitor at concentrations above 500 nM, compatibly with residual activity in this mutant. The study highlights the need for extending plant innate immune response studies to assays sampling EFR signaling at the molecular level.
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http://dx.doi.org/10.3390/genes10010015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357087PMC
December 2018

In Planta Preliminary Screening of ER Glycoprotein Folding Quality Control (ERQC) Modulators.

Int J Mol Sci 2018 Jul 23;19(7). Epub 2018 Jul 23.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Small molecule modulators of the Endoplasmic Reticulum glycoprotein folding quality control (ERQC) machinery have broad-spectrum antiviral activity against a number of enveloped viruses and have the potential to rescue secretion of misfolded but active glycoproteins in rare diseases. In vivo assays of candidate inhibitors in mammals are expensive and cannot be afforded at the preliminary stages of drug development programs. The strong conservation of the ERQC machinery across eukaryotes makes transgenic plants an attractive system for low-cost, easy and fast proof-of-concept screening of candidate ERQC inhibitors. The immune response is mediated by glycoproteins, the folding of which is controlled by ERQC. We have used the plant response to bacterial peptides as a means of assaying an ERQC inhibitor in vivo. We show that the treatment of the plant with the iminosugar B-DNJ, which is a known ER α-glucosidase inhibitor in mammals, influences the immune response of the plant to the bacterial peptide elf18 but not to the flagellin-derived flg22 peptide. In the B-DNJ-treated plant, the responses to elf18 and flg22 treatments closely follow the ones observed for the ER α-glucosidase II impaired plant, . We propose as a promising platform for the development of low-cost proof-of-concept in vivo ERQC modulation.
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http://dx.doi.org/10.3390/ijms19072135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073501PMC
July 2018

Structural basis of pyrrole polymerization in human porphobilinogen deaminase.

Biochim Biophys Acta Gen Subj 2018 09 15;1862(9):1948-1955. Epub 2018 Jun 15.

Protein Stability and Inherited Disease Laboratory, CIC bioGUNE, Derio, Bizkaia 48160, Spain. Electronic address:

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.
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http://dx.doi.org/10.1016/j.bbagen.2018.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192514PMC
September 2018

Structural Insights into the Broad-Spectrum Antiviral Target Endoplasmic Reticulum Alpha-Glucosidase II.

Adv Exp Med Biol 2018;1062:265-276

Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, UK.

Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and β-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.
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http://dx.doi.org/10.1007/978-981-10-8727-1_19DOI Listing
November 2018

Structural basis of cholesterol binding by a novel clade of dendritic cell modulators from ticks.

Sci Rep 2017 11 22;7(1):16057. Epub 2017 Nov 22.

Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, England, United Kingdom.

Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.
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http://dx.doi.org/10.1038/s41598-017-16413-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700055PMC
November 2017

Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016).

Proteins 2018 03 16;86 Suppl 1:27-50. Epub 2017 Oct 16.

Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (UMR 5280 - CNRS, ENS Lyon, UCB Lyon 1), Villeurbanne, 69100, France.

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.
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http://dx.doi.org/10.1002/prot.25392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820184PMC
March 2018

Anharmonic motions versus dynamic disorder at the Mg ion from the charge densities in pyrope (MgAlSiO) crystals at 30 K: six of one, half a dozen of the other.

Acta Crystallogr B Struct Sci Cryst Eng Mater 2017 Aug 25;73(Pt 4):722-736. Epub 2017 Jul 25.

Department of Chemistry, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.

The possible occurrence of static/dynamic disorder at the Mg site in pyrope (MgAlSiO), with or without anharmonic contribution to the thermal vibrations even at low temperatures, has been largely debated but conclusions were contrasting. Here a report is given on the experimental charge density distribution, ρ, of synthetic pyrope at T = 30 K, built through a Stewart multipolar expansion up to l = 5 and based on a very precise and accurate set of in-home measured single-crystal X-ray diffraction amplitudes with a maximum resolution of 0.44 Å. Local and integral topological properties of ρ are in substantial agreement with those of ρ, the corresponding DFT-grade quantum charge density of an ideal pyrope crystal, and those derived from synchrotron investigations of chemical bonding in olivines. Relevant thermal atomic displacements, probably anharmonic in nature, clearly affect the whole structure down to 30 K. No significant (> 2.5σ) residual Fourier peaks are detectable from the ρ distribution around Mg, after least-squares refinement of a multipole model with anharmonic thermal motion at the Mg site. Experimental findings were confirmed by a full analysis of normal vibration modes of the DFT-optimized structure of the perfect pyrope crystal. Mg undergoes wide displacements from its equilibrium position even at very low temperatures, as it is allocated in a ∼ 4.5 Å large dodecahedral cavity and involved in several soft phonon modes. Implications on the interplay among static/dynamic disorder of Mg and lattice vibrational degrees of freedom are discussed.
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http://dx.doi.org/10.1107/S2052520617006102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181205PMC
August 2017

Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint.

Proc Natl Acad Sci U S A 2017 08 24;114(32):8544-8549. Epub 2017 Jul 24.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.
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http://dx.doi.org/10.1073/pnas.1703682114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559018PMC
August 2017

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.

Nat Commun 2017 05 2;8:15075. Epub 2017 May 2.

Department of Molecular and Cellular Biochemistry, Indiana University, Simon Hall MSB, 212 S Hawthorne Dr, Bloomington, Indiana 47405, USA.

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
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http://dx.doi.org/10.1038/ncomms15075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418573PMC
May 2017

Mechanism of Structural Tuning of the Hepatitis C Virus Human Cellular Receptor CD81 Large Extracellular Loop.

Structure 2017 01 1;25(1):53-65. Epub 2016 Dec 1.

Structural Biology Unit, CIC bioGUNE, CIBERehd, Derio 48160, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao 48013, Spain. Electronic address:

Hepatitis C virus (HCV) enters into human hepatocytes via tetraspanin hCD81. HCV glycoprotein E2 recognizes the "head" subdomain of the large extracellular loop (LEL) of CD81 (hCD81), but the precise mechanism of virus cell attachment and entry remains elusive. Here, by combining the structural analysis of a conspicuous number of crystallized CD81 molecules with molecular dynamics simulations, we show that the conformational plasticity of the hCD81 head subdomain is a molecular property of the receptor. The observed closed, intermediate, and open conformations of the head subdomain provide distinct binding platforms. Simulations at pH 7.4 and 4.0 indicate that this dynamism is pH modulated. The crystallized double conformation of the disulfide bridge C157-C175 at the base of the head subdomain identifies this bond as the molecular zipper of the plasticity of hCD81. We propose that this conformational dependence of hCD81, which is finely tuned by pH and redox conditions, enables the virus-receptor interactions to diversely re-engage at endosomal conditions.
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http://dx.doi.org/10.1016/j.str.2016.11.003DOI Listing
January 2017

Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals.

Proc Natl Acad Sci U S A 2016 08 26;113(32):E4630-8. Epub 2016 Jul 26.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;

The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.
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http://dx.doi.org/10.1073/pnas.1604463113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987793PMC
August 2016

Super-complexes of adhesion GPCRs and neural guidance receptors.

Nat Commun 2016 Apr 19;7:11184. Epub 2016 Apr 19.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK.

Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger 'super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.
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http://dx.doi.org/10.1038/ncomms11184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838878PMC
April 2016

Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding.

Elife 2015 Apr 10;4:e05345. Epub 2015 Apr 10.

Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation.
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http://dx.doi.org/10.7554/eLife.05345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420994PMC
April 2015

Structural basis of latrophilin-FLRT interaction.

Structure 2015 Apr 26;23(4):774-81. Epub 2015 Feb 26.

Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

Latrophilins, receptors for spider venom α-latrotoxin, are adhesion type G-protein-coupled receptors with emerging functions in synapse development. The N-terminal region binds the endogenous cell adhesion molecule FLRT, a major regulator of cortical and synapse development. We present crystallographic data for the mouse Latrophilin3 lectin and olfactomedin-like (Olf) domains, thereby revealing the Olf β-propeller fold and conserved calcium-binding site. We locate the FLRT-Latrophilin binding surfaces by a combination of sequence conservation analysis, point mutagenesis, and surface plasmon resonance experiments. In stripe assays, we show that wild-type Latrophilin3 and its high-affinity interactor FLRT2, but not the binding-impaired mutants we generated, promote HeLa cell adhesion. In contrast, cortical neurons expressing endogenous FLRTs are repelled by wild-type Latrophilin3 and not by the binding-impaired mutant. Taken together, we present molecular level insights into Latrophilin structure, its FLRT-binding mechanism, and a role for Latrophilin and FLRT that goes beyond a simply adhesive interaction.
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http://dx.doi.org/10.1016/j.str.2015.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396693PMC
April 2015

The Black cells phenotype is caused by a point mutation in the Drosophila pro-phenoloxidase 1 gene that triggers melanization and hematopoietic defects.

Dev Comp Immunol 2015 Jun 24;50(2):166-74. Epub 2014 Dec 24.

Global Health Institute, Swiss Federal Institute of Technology, Station 19, CH-1015 Lausanne, Switzerland. Electronic address:

Melanization contributes to arthropod-specific innate immunity through deposition of melanin at wound sites or around parasites, with concomitant release of microbicidal reactive oxygen species. Melanization requires sequential activation of proteolytic enzymes in the hemolymph, including the final enzyme pro-phenoloxidase. Black cells (Bc) is a mutation causing spontaneous melanization of Drosophila crystal cells, a hemocyte cell type producing phenoloxidases. Bc individuals exhibit circulating black spots but fail to melanize upon injury. Although Bc is widely used as a loss-of-function mutant of phenoloxidases, the mutation causing Bc remained unknown. Here, we identified a single point mutation in the pro-phenoloxidase 1 (PPO1) gene of Bc flies causing an Alanine to Valine change in the C-terminal domain of PPO1, predicted to affect the conformation of the N-terminal pro-domain cleavage site at a distance and causing uncontrolled catalytic activity. Genomic insertion of a PPO1(A480V) transgene phenocopies Black cells, proving that A480V is indeed the causal mutation of the historical Bc phenotype.
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http://dx.doi.org/10.1016/j.dci.2014.12.011DOI Listing
June 2015

A complex iron-calcium cofactor catalyzing phosphotransfer chemistry.

Science 2014 Sep;345(6201):1170-1173

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.

Alkaline phosphatases play a crucial role in phosphate acquisition by microorganisms. To expand our understanding of catalysis by this class of enzymes, we have determined the structure of the widely occurring microbial alkaline phosphatase PhoX. The enzyme contains a complex active-site cofactor comprising two antiferromagnetically coupled ferric iron ions (Fe(3+)), three calcium ions (Ca(2+)), and an oxo group bridging three of the metal ions. Notably, the main part of the cofactor resembles synthetic oxide-centered triangular metal complexes. Structures of PhoX-ligand complexes reveal how the active-site metal ions bind substrate and implicate the cofactor oxo group in the catalytic mechanism. The presence of iron in PhoX raises the possibility that iron bioavailability limits microbial phosphate acquisition.
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http://dx.doi.org/10.1126/science.1254237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175392PMC
September 2014

Investigating the structure of the factor B vWF-A domain/CD55 protein-protein complex using DEER spectroscopy: successes and pitfalls.

Mol Phys 2013 Oct 9;111(18-19):2865-2872. Epub 2013 Oct 9.

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

The electron paramagnetic resonance technique of double electron-electron resonance (DEER) was used to measure nanometre-scale distances between nitroxide spin labels attached to the complement regulatory protein CD55 (also known as decay accelerating factor) and the von Willebrand factor A (vWF-A) domain of factor B. Following a thorough assessment of the quality of the data, distances obtained from good-quality measurements are compared to predicted distances from a previously hypothesised model for the complex and are found to be incompatible. The success of using these distances as restraints in multi-body docking routines is presented critically.
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http://dx.doi.org/10.1080/00268976.2013.827754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056885PMC
October 2013

Identification and characterization of the 'missing' terminal enzyme for siroheme biosynthesis in α-proteobacteria.

Mol Microbiol 2014 Apr 13;92(1):153-63. Epub 2014 Mar 13.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

It has recently been shown that the biosynthetic route for both the d1 -haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1 -haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.
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http://dx.doi.org/10.1111/mmi.12542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063343PMC
April 2014

Molecular replacements.

Acta Crystallogr D Biol Crystallogr 2013 Nov 18;69(Pt 11):2165-6. Epub 2013 Oct 18.

Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK.

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http://dx.doi.org/10.1107/S0907444913027352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817688PMC
November 2013

Coronary artery disease concomitant with chronic obstructive pulmonary disease.

Eur J Clin Invest 2014 Jan 26;44(1):93-102. Epub 2013 Oct 26.

Section of Cardiology, Department of Medicine and Emergency Medicine, University of Modena and Reggio Emilia, Policlinico di Modena, Modena, Italy.

Background: Numerous epidemiologic studies have linked the presence of chronic obstructive pulmonary disease (COPD) to coronary artery disease (CAD). However, prevalence, pathological processes, clinical manifestations and therapy are still debated, as progress towards uncovering the link between these two disorders has been hindered by the complex nature of multimorbidity.

Methods: Articles targeting CAD in patients with COPD were identified from the searches of MEDLINE and EMBASE databases in July 2013. Three authors reviewed available evidence, focusing on the latest development on disease prevalence, pathogenesis, clinical manifestations and therapeutic strategies. Both clinical trial and previous reviews have been included in this work.

Results: The most accredited hypothesis asserts that the main common risk factors, that is, cigarette smoke and ageing, elicit a chronic low-grade systemic inflammatory response, which affects both cardiovascular endothelial cells and airways/lung parenchyma. The development of CAD in patients with COPD potentiates the morbidity of COPD, leading to increased hospitalizations, mortality and health costs. Moreover, correct diagnosis is challenging and therapies are not clearly defined.

Conclusions: Evidence from recently published articles highlights the importance of multimorbidity in patient management and future research. Moreover, many authors emphasize the importance of low-grade systemic inflammation as a common pathological mechanism and a possible future therapeutic target.
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http://dx.doi.org/10.1111/eci.12181DOI Listing
January 2014
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