Enzymes with radical tendencies : the PFL family

The first glycyl radical in an enzyme was described 20 years ago and since then the family of glycyl radical enzymes (GREs) has expanded to include enzymes catalysing five chemically distinct reactions. The type enzymes of the family, anaerobic ribonucleotide reductase (RNRIII) and pyruvate formate lyase (PFL) had been studied long before it was known that they are GREs. Spectroscopic measurements on the radical and an observation that exposure to oxygen irreversibly inactivates the enzymes by cleavage of the protein proved that the radical is located on a particular glycine residue, close to the C-terminus of the protein. Both anaerobic RNRIII and PFL, are important for many anaerobic and facultative anaerobic bacteria as RNRIII is responsible for the synthesis of DNA precursors and PFL catalyses a key metabolic reaction in glycolysis. The crystal structures of both were solved in 1999 and they revealed that, although the enzymes do not share significant sequence identity, they share a similar structure - the radical site and residues necessary for catalysis are buried inside a ten stranded \ualpha /\ubeta -barrel. GREs are synthesised in an inactive form and are post-translationally activated by an activating enzyme which uses S-adenosyl methionine and an iron-sulphur cluster to generate the radical. One of the goals of this thesis work was to crystallise the activating enzyme of PFL. This task is challenging as, like GREs, the activating component is inactivated by oxygen. The experiments were therefore carried out in an oxygen free atmosphere. This is the first report of a crystalline GRE activating enzyme. Recently several new GREs have been characterised, all sharing sequence similarity to PFL but not to RNRIII. Also, the genome sequencing projects have identified many PFL-like GREs of unknown function, usually annotated as PFLs. In the present thesis I describe the grouping of these PFL family enzymes based on the sequence similarity and analyse the conservation patterns when compared to the structure of E. coli PFL. Based on this information an activation route is proposed. I also report a crystal structure of one of the PFL-like enzymes with unknown function, PFL2 from Archaeoglobus fulgidus. As A. fulgidus is a hyperthermophilic organism, possible mechanisms stabilising the structure are discussed. The organisation of an active site of PFL2 suggests that the enzyme may be a dehydratase. Keywords: glycyl radical, enzyme, pyruvate formate lyase, x-ray crystallography, bioinformaticsGlysyyliradikaalientsyymit (GRE:t) muodostavat proteiiniperheen, jonka jäsenet aktivoiduttuaan varastoivat radikaalin glysiini aminohappoon. GRE:t toimivat ainoastaan anaerobisissa eli hapettomissa olosuhteissa. Jos nämä entsyymit altistuvat hapelle, niiden aminohappoketju katkeaa ja entsyymi inaktivoituu. Eniten tutkitut GRE:t, anaerobinen ribonukleotidireduktaasi (RNRIII) ja pyruvaattiformaattilyaasi (PFL), ovat tietyille hapettomissa oloissa eläville organismeille välttämättömiä entsyymejä. RNRIII katalysoi reaktiota jota tarvitaan DNA:n valmistukseen. PFL puolestaan toimii mikrobin anaerobisessa energiantuotannossa. Sekä RNRIII:n että PFL:n rakenteet on ratkaistu ja ne muistuttavat toisiaan, vaikka niiden aminohappojärjestys on hyvin erilainen. Väitöskirjatyössäni olen tutkinut GRE:ejä röntgensädekristallografian ja bioinformatiikan menetelmin. PFL:n katalysoimassa reaktiossa pyruvaatin hiiliatomien välinen sidos katkeaa ja syntyy lopulta formaattia ja asetyylikoentsyymi A:ta. PFL:n kiderakenne yhdessä pyruvaatin kanssa paljasti, että pyruvaatti sitoutuu aktiiviseen keskukseen hyvin samalla tavalla kuin sen analogi oksamaatti. Oksamaatti on kuitenkin inertti, eikä toimi entsyymin varsinaisena substraattina eikä inhibiittorina. Onkin todennäköistä, että entsyymin aktivoiduttua eli radikaalin muodostumisen jälkeen, aktiivisen keskuksen konformaatio on muuttunut. Olisi tärkeää selvittää aktivoidun entsyymin kiderakenne, jotta rakenteen perusteella voitaisiin tehdä johtopäätöksiä entsyymin toimintamekanismista. Viime vuosien aikana tieto eri organismien perimästä on kasvanut valtavasti ja sen johdosta on löydetty uusia, emässekvenssin perusteella GRE:n kaltaisia geenejä. Väitöskirjatyössä vertailtiin tietokannoista löytyvien GRE:n kaltaisten entsyymien aminohappojärjestyksiä, ryhmiteltiin ne samankaltaisiin joukkoihin ja pyrittiin löytämään vakioisia alueita eri joukkojen sisältä sekä niiden väliltä. Saatua tietoa verrattiin aiemmin ratkaistuun PFL:n kiderakenteeseen. Vakioisten alueiden perusteella ehdotettiin mm. entsyymin aktivointireittiä, jonka kautta GRE:ä aktivoiva entsyymi pystyisi luomaan glysyyliradikaalin. Tietokannoista löytyvistä sekvensseistä löydettiin myös uusi aiemmin tunnistamaton GRE ryhmä, jonka jäsenet ovat merkittävästi pienempiä kuin aiemmin tunnetut entsyymit. Yksi tuntematon entsyymi, arkkibakteerin PFL2, valittiin tarkemman tutkimuksen kohteeksi, koska kyseinen arkkibakteeri, Archaeoglobus fulgidus, elää hyvin korkeissa lämpötiloissa, parhaiten 83 asteessa. A. fulgiduksen epätavallinen kasvulämpötila asettaa erityisvaatimuksia myös sen proteiineille, sillä näin korkeassa lämpötilassa proteiinit yleensä denaturoituvat. Korkean lämpökestävyyden takia PFL2 entsyymin glysyyliradikaalin voidaan olettaa olevan huoneenlämmössä stabiilimpi kuin PFL:n vastaava. Tämä saattaa mahdollistaa aktiivisen entsyymin rakenteen tutkimisen. Yhdistelmä-DNA-tekniikan avulla A. fulgiduksesta monistettiin PFL2:ta koodaava geeni ja tuotettiin entsyymiä Escherichia coli-bakteerissa. Proteiini puhdistettiin, kiteytettiin ja sen rakenne ratkaistiin röntgensädekristallografian avulla. PFL2:n rakenne muistuttaa hyvin paljon yllä mainitun PFL:n rakennetta, mutta se on kuitenkin hieman lähempänä toisen GRE:n, hiljattain löydetyn glyseroli dehydrataasin (GD), rakennetta. PFL2:n kiderakenteessa aktiiviseen keskukseen on sitoutuneena pienmolekyyli, jonka elektronitiheys vastaa glyserolia. PFL2:n ja GD:n kiderakenteiden perusteella vaikuttaisi, että PFL2 olisi myös dehydrataasi, jonka substraatti on glyserolin kaltainen - ehkä hieman suurempi. Muista GRE:istä poiketen PFL2 on tetrameeri, joka koostuu kahden sijaan neljästä samanlaisesta alayksiköstä. Tällä on merkitystä entsyymin säätelyn kannalta, sillä yleensä ainoastaan toinen GRE:n alayksiköistä on aktiivinen. PFL2:n rakenteesta löytyi useita tekijöitä, jotka mahdollistavat entsyymin korkean lämpökestävyyden: mm. parempi aminohappojen pakkautuminen, suurempi määrä varauspareja, lyhyemmät silmukat ja niiden ankkurointi sekä ehkä myös suurempi alayksikköjen määrä. Avainsanat: glysyyliradikaali, entsyymi, pyruvaattiformaattilyaasi, röntgensädekristallografia, bioinformatiikk

Inhibitor screening assay for neurexin-LRRTM adhesion protein interaction involved in synaptic maintenance and neurological disorders.

Synaptic adhesion molecules, including presynaptic neurexins (NRXNs) and post-synaptic leucine-rich repeat transmembrane (LRRTM) proteins are important for development and maintenance of brain neuronal networks. NRXNs are probably the best characterized synaptic adhesion molecules, and one of the major presynaptic organizer proteins. The LRRTMs were found as ligands for NRXNs. Many of the synaptic adhesion proteins have been linked to neurological cognitive disorders, such as schizophrenia and autism spectrum disorders, making them targets of interest for both biological studies, and towards drug development. Therefore, we decided to develop a screening method to target the adhesion proteins, here the LRRTM-NRXN interaction, to find small molecule probes for further studies in cellular settings. To our knowledge, no potent small molecule compounds against the neuronal synaptic adhesion proteins are available. We utilized the AlphaScreen technology, and developed an assay targeting the NRXN-LRRTM2 interaction. We carried out screening of 2000 compounds and identified hits with moderate IC50-values. We also established an orthogonal in-cell Western blot assay to validate hits. This paves way for future development of specific high affinity compounds by further high throughput screening of larger compound libraries using the methods established here. The method could also be applied to screening other NRXN-ligand interactions.Peer reviewe

Macrodomain binding compound MRS 2578 inhibits alphavirus replication

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Macrodomain binding compound MRS 2578 inhibits alphavirus replication

Peer reviewe

Completing the family portrait of the anti-apoptotic Bcl-2 proteins: Crystal structure of human Bfl-1 in complex with Bim

AbstractEvasion of apoptosis is recognized as a characteristic of malignant growth. Anti-apoptotic B-cell lymphoma-2 (Bcl-2) family members have therefore emerged as potential therapeutic targets due to their critical role in proliferating cancer cells. Here, we present the crystal structure of Bfl-1, the last anti-apoptotic Bcl-2 family member to be structurally characterized, in complex with a peptide corresponding to the BH3 region of the pro-apoptotic protein Bim. The structure reveals distinct features at the peptide-binding site, likely to define the binding specificity for pro-apoptotic proteins. Superposition of the Bfl-1:Bim complex with that of Mcl-1:Bim reveals a significant local plasticity of hydrophobic interactions contributed by the Bim peptide, likely to be the basis for the multi specificity of Bim for anti-apoptotic proteins

The Crystal Structure of the Dachshund Domain of Human SnoN Reveals Flexibility in the Putative Protein Interaction Surface

The human SnoN is an oncoprotein that interacts with several transcription-regulatory proteins such as the histone-deacetylase, N-CoR containing co-repressor complex and Smad proteins. This study presents the crystal structure of the Dachshund homology domain of human SnoN. The structure reveals a groove composed of conserved residues with characteristic properties of a protein-interaction surface. A comparison of the 12 monomers in the asymmetric unit reveals the presence of two major conformations: an open conformation with a well accessible groove and a tight conformation with a less accessible groove. The variability in the backbone between the open and the tight conformations matches the differences seen in previously determined structures of individual Dachshund homology domains, suggesting a general plasticity within this fold family. The flexibility observed in the putative protein binding groove may enable SnoN to recognize multiple interaction partners

Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei

TbPARG in Trypanosoma brucei. A) TbPARG localization in untreated (control) and in procyclic cultures exposed to 500 μM H2O2 for 10 min. IFI was carried out as reported in our previous work [33]. TbPARG was identified with our home-made antibody against TcPARG [33]; and PAR was identified with a commercial antibody against PAR (BD). White bar represents 50 μm. B) Western blot analysis of 40 μg protein per lane revealed with a commercial anti-PARG antibody (Antibody Verify) in T. brucei procyclic (PC) and bloodstream (BST) forms. The arrow indicates the band with the expected molecular weight (approximately 60 kDa). The membrane stained with Red Ponceau was used as a loading control. (TIF 4272 kb

Zinc Binding Catalytic Domain of Human Tankyrase 1

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substratebinding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular

Adenosine analogs bearing phosphate isosteres as human MDO1 ligands

The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 50-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations. (C) 2018 Elsevier Ltd. All rights reserved.Peer reviewe

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARSCoV- 2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFNresponsive genes. Our results suggest that PARP9/DTX3Ldependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARSCoV- 2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself