Structural studies on Porphyromonas gingivalis proteins

[eng] We investigated the structure function relationship of PorX by combined biophysical experiments and enzymatic assays. As a regulator of the type 9 secretion system (T9SS) of the human pathogen Porphyromonas gingivalis (PG), PorX is responsible for PG virulence[1] and as drug target could be th...

Descripción completa

Detalles Bibliográficos
Autor: Schmitz, Claus-A.
Tipo de recurso: tesis doctoral
Estado:Versión publicada
Fecha de publicación:2023
País:España
Institución:Universidad de Barcelona
Repositorio:Dipòsit Digital de la UB
OAI Identifier:oai:diposit.ub.edu:2445/203352
Acceso en línea:https://hdl.handle.net/2445/203352
http://hdl.handle.net/10803/689250
Access Level:acceso abierto
Palabra clave:Bacteris anaerobis
Radiocristal·lografia
Malalties periodontals
Anaerobic bacteria
X-ray crystallography
Periodontal disease
id ES_cfeb736eab474049c4b671c3e134f8a0
oai_identifier_str oai:diposit.ub.edu:2445/203352
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Structural studies on Porphyromonas gingivalis proteins
title Structural studies on Porphyromonas gingivalis proteins
spellingShingle Structural studies on Porphyromonas gingivalis proteins
Schmitz, Claus-A.
Bacteris anaerobis
Radiocristal·lografia
Malalties periodontals
Anaerobic bacteria
X-ray crystallography
Periodontal disease
title_short Structural studies on Porphyromonas gingivalis proteins
title_full Structural studies on Porphyromonas gingivalis proteins
title_fullStr Structural studies on Porphyromonas gingivalis proteins
title_full_unstemmed Structural studies on Porphyromonas gingivalis proteins
title_sort Structural studies on Porphyromonas gingivalis proteins
dc.creator.none.fl_str_mv Schmitz, Claus-A.
author Schmitz, Claus-A.
author_facet Schmitz, Claus-A.
author_role author
dc.contributor.none.fl_str_mv Solà Vilarrubias, Maria
Universitat de Barcelona. Facultat de Farmàcia i Ciències de l'Alimentació
dc.subject.none.fl_str_mv Bacteris anaerobis
Radiocristal·lografia
Malalties periodontals
Anaerobic bacteria
X-ray crystallography
Periodontal disease
topic Bacteris anaerobis
Radiocristal·lografia
Malalties periodontals
Anaerobic bacteria
X-ray crystallography
Periodontal disease
description [eng] We investigated the structure function relationship of PorX by combined biophysical experiments and enzymatic assays. As a regulator of the type 9 secretion system (T9SS) of the human pathogen Porphyromonas gingivalis (PG), PorX is responsible for PG virulence[1] and as drug target could be the key in disease prevention. We started with crystals of wild type PorX dimers, which were unsuitable for structure solution as well as further PG proteins for potential cocrystallization. We successfully optimized the crystallization conditions for selenomethionine derivative PorX by adding beryllium fluoride as phosphate mimic, zinc for higher occupancies in the active site, as well as additives and optimizing buffers, concentrations, crystallization- and crystal manipulation techniques. By X-ray diffraction and single anomalous dispersion we solved several structures of dimeric PorX with and without substrate (pGpG) up to a resolution of 1.9 Å, characterized its domains, active site and substrate binding pocket. The PorX N-terminal domain is a typical response regulator receiver domain (RD) of the OmpR[2] subfamily. PorX has the characteristic α5β5 Rossmann-like fold, conserved phosphorylation site and the YT pair responsible for conformational change during phosphorylation which leads to dimerization. A helical bundle domain (HBD) connects the RD to the C-terminal enzymatic active PglZ domain which was previously uncharacterized in structure and function. The PglZ domain has the characteristic α/β-fold of the alkaline phosphatase superfamily (APS), the metal coordination sphere of phosphodiesterase, and an extra domain capping the active site (CAP[3]).[4], [5] By small-angle X-ray scattering we investigated PorX dimers in solution, which did not significantly differed from the crystal structure, and PorX monomers showing significant conformational changes. We build and validated a SAXS model of monomeric PorX with a χ² of 1.15 and explained the mechanism of PorX dimerization: Phosphorylation leads to RD dimerization, their conformational changes lead to an exchange of surfaces between RDs and HBDs, which then bring both PglZs into contact, followed by PglZ dimerization. The newly formed PglZ dimeric interface (DI) folds into the catalytic active APS bi-metallo active site, and the capping subdomain opens the substrate binding pocket due to interaction with DI. These we confirmed by structure based mutational studies, SEC-MALLS as well as phosphorylation assays, SEC and enzymatic assays performed by our collaboration partners. Comparing PglZ subfamily homologs, we found out that the active center as well as its DI is conserved with most BREX-PglZs, involved in phage defense[6]. In PorX the DI folds upon dimerization and thus activates the protein, which thereby is likely also true for BREX-PglZs. Our collaborators determined the enzymatic activity by enzymatic assays confirming PorX as phosphodiesterase and not a phosphatase even though APS active sites often have promiscuous activity[7], [8]. Phosphodiesterase substrate affinities are regulated by the substrate binding pockets, so in order to narrow down potential substrates to screen, we compared PorX to its closest known structures and enzyme classes. As results we found the most similar substrate cavities in ecto- nucleotide pyrophosphatases which cleave linear and cyclic dinucleotides as well as mono- and dinucleotide polyphosphates. This led to cocrystallization trials yielding our pGpG bound structure with the PorX substrate binding pocket only partial filled. This led us screen activity against a large polynucleotide library and product detection using ultra- high performance liquid-chromatography, coupled with mass spectrometry (UHPLC-MS). As result PorX cleaved linear and cyclic oligonucleotides as well as linear dinucleotides. We did not found any activity against cyclic mono- or dinucleotides, mono- or dinucleotide polyphosphates. Therefore it is likely that PorX is involved in oligonucleotide signaling in PG and by designing of PorX PglZ inhibitors it might be possible to stop PG T9SS transcriptional activation and render PG avirulent. Summary Literature 1: Veith, Paul D.; Glew, Michelle D.; Gorasia, Dhana G.; Reynolds, Eric C., "Type IX secretion", Molecular microbiology 106, 1, p35–53, 2017. 2: Gao, Rong; Bouillet, Sophie; Stock, Ann M., Structural Basis of Response Regulator Function, 2019 3: Kim, A; Benning, MM; OkLee, S; Quinn, J Martin, BM; Holden, HM; Dunaway-Mariano, D, "Divergence of chemical function in the alkaline phosphatase superfamily", Biochemistry 50, 17, p3481–3494, 2011. 4: Galperin, Michael Y.; Koonin, Eugene V., "Divergence and convergence in enzyme evolution", The Journal of biological chemistry 287, 1, p21–28, 2012. 5: Sunden F, AlSadhan I, Lyubimov A, Doukov T, Swan J, Herschlag D., "Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.", J Biol Chem. 292, 51, p20960–20974, 2017. 6: Chaudhary, Kulbhushan, "BacteRiophage EXclusion (BREX)", Journal of cellular physiology 233, 2, p771– 773, 2018. 7: Mohamed, MF.; Hollfelder, F., "Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer", Biochimica et biophysica acta 1834, 1, p417–424, 2013. 8: Pabis, Anna; Kamerlin, Shina Caroline Lynn, "Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily", Current opinion in structural biology 37, p14–21, 2016.
publishDate 2023
dc.date.none.fl_str_mv 2023
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
info:eu-repo/semantics/publishedVersion
format doctoralThesis
status_str publishedVersion
dc.identifier.none.fl_str_mv https://hdl.handle.net/2445/203352
http://hdl.handle.net/10803/689250
url https://hdl.handle.net/2445/203352
http://hdl.handle.net/10803/689250
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv (c) Schmitz, Claus-A., 2023
info:eu-repo/semantics/openAccess
rights_invalid_str_mv (c) Schmitz, Claus-A., 2023
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universitat de Barcelona
publisher.none.fl_str_mv Universitat de Barcelona
dc.source.none.fl_str_mv Tesis Doctorals - Facultat - Farmàcia i Ciències de l'Alimentació
reponame:Dipòsit Digital de la UB
instname:Universidad de Barcelona
instname_str Universidad de Barcelona
reponame_str Dipòsit Digital de la UB
collection Dipòsit Digital de la UB
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869420126646829056
spelling Structural studies on Porphyromonas gingivalis proteinsSchmitz, Claus-A.Bacteris anaerobisRadiocristal·lografiaMalalties periodontalsAnaerobic bacteriaX-ray crystallographyPeriodontal disease[eng] We investigated the structure function relationship of PorX by combined biophysical experiments and enzymatic assays. As a regulator of the type 9 secretion system (T9SS) of the human pathogen Porphyromonas gingivalis (PG), PorX is responsible for PG virulence[1] and as drug target could be the key in disease prevention. We started with crystals of wild type PorX dimers, which were unsuitable for structure solution as well as further PG proteins for potential cocrystallization. We successfully optimized the crystallization conditions for selenomethionine derivative PorX by adding beryllium fluoride as phosphate mimic, zinc for higher occupancies in the active site, as well as additives and optimizing buffers, concentrations, crystallization- and crystal manipulation techniques. By X-ray diffraction and single anomalous dispersion we solved several structures of dimeric PorX with and without substrate (pGpG) up to a resolution of 1.9 Å, characterized its domains, active site and substrate binding pocket. The PorX N-terminal domain is a typical response regulator receiver domain (RD) of the OmpR[2] subfamily. PorX has the characteristic α5β5 Rossmann-like fold, conserved phosphorylation site and the YT pair responsible for conformational change during phosphorylation which leads to dimerization. A helical bundle domain (HBD) connects the RD to the C-terminal enzymatic active PglZ domain which was previously uncharacterized in structure and function. The PglZ domain has the characteristic α/β-fold of the alkaline phosphatase superfamily (APS), the metal coordination sphere of phosphodiesterase, and an extra domain capping the active site (CAP[3]).[4], [5] By small-angle X-ray scattering we investigated PorX dimers in solution, which did not significantly differed from the crystal structure, and PorX monomers showing significant conformational changes. We build and validated a SAXS model of monomeric PorX with a χ² of 1.15 and explained the mechanism of PorX dimerization: Phosphorylation leads to RD dimerization, their conformational changes lead to an exchange of surfaces between RDs and HBDs, which then bring both PglZs into contact, followed by PglZ dimerization. The newly formed PglZ dimeric interface (DI) folds into the catalytic active APS bi-metallo active site, and the capping subdomain opens the substrate binding pocket due to interaction with DI. These we confirmed by structure based mutational studies, SEC-MALLS as well as phosphorylation assays, SEC and enzymatic assays performed by our collaboration partners. Comparing PglZ subfamily homologs, we found out that the active center as well as its DI is conserved with most BREX-PglZs, involved in phage defense[6]. In PorX the DI folds upon dimerization and thus activates the protein, which thereby is likely also true for BREX-PglZs. Our collaborators determined the enzymatic activity by enzymatic assays confirming PorX as phosphodiesterase and not a phosphatase even though APS active sites often have promiscuous activity[7], [8]. Phosphodiesterase substrate affinities are regulated by the substrate binding pockets, so in order to narrow down potential substrates to screen, we compared PorX to its closest known structures and enzyme classes. As results we found the most similar substrate cavities in ecto- nucleotide pyrophosphatases which cleave linear and cyclic dinucleotides as well as mono- and dinucleotide polyphosphates. This led to cocrystallization trials yielding our pGpG bound structure with the PorX substrate binding pocket only partial filled. This led us screen activity against a large polynucleotide library and product detection using ultra- high performance liquid-chromatography, coupled with mass spectrometry (UHPLC-MS). As result PorX cleaved linear and cyclic oligonucleotides as well as linear dinucleotides. We did not found any activity against cyclic mono- or dinucleotides, mono- or dinucleotide polyphosphates. Therefore it is likely that PorX is involved in oligonucleotide signaling in PG and by designing of PorX PglZ inhibitors it might be possible to stop PG T9SS transcriptional activation and render PG avirulent. Summary Literature 1: Veith, Paul D.; Glew, Michelle D.; Gorasia, Dhana G.; Reynolds, Eric C., "Type IX secretion", Molecular microbiology 106, 1, p35–53, 2017. 2: Gao, Rong; Bouillet, Sophie; Stock, Ann M., Structural Basis of Response Regulator Function, 2019 3: Kim, A; Benning, MM; OkLee, S; Quinn, J Martin, BM; Holden, HM; Dunaway-Mariano, D, "Divergence of chemical function in the alkaline phosphatase superfamily", Biochemistry 50, 17, p3481–3494, 2011. 4: Galperin, Michael Y.; Koonin, Eugene V., "Divergence and convergence in enzyme evolution", The Journal of biological chemistry 287, 1, p21–28, 2012. 5: Sunden F, AlSadhan I, Lyubimov A, Doukov T, Swan J, Herschlag D., "Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.", J Biol Chem. 292, 51, p20960–20974, 2017. 6: Chaudhary, Kulbhushan, "BacteRiophage EXclusion (BREX)", Journal of cellular physiology 233, 2, p771– 773, 2018. 7: Mohamed, MF.; Hollfelder, F., "Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer", Biochimica et biophysica acta 1834, 1, p417–424, 2013. 8: Pabis, Anna; Kamerlin, Shina Caroline Lynn, "Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily", Current opinion in structural biology 37, p14–21, 2016.[ger] In dieser Doktorarbeit beschreiben wir die Struktur und Funktion von PorX, einem essentiellen Protein des Typ-9-Sekretionssystems von Porphyromaonas gingivalis (PG).[1] PG ist Teil des dentalen Biofilms und gilt als Hauptauslöser für Gingivitis und Parodontitis.[2] PG befällt nicht nur orales Gewebe, sondern befällt auch u.a. Gehirn, Leber und Koronararterien. [3], [4], [5] Es ist an einer Vielzahl von systemischen Erkrankungen beteiligt, darunter Krebs[6] Arteriosklerose[7], [8] und Herzkreislauf-erkrankungen.[9] [10] Da die Gesamtheit dieser Krankheiten die Mehrheit der menschlichen Bevölkerung betrifft ist die Erforschung von PorX ein lohnendes Forschungsprojekt, nicht zuletzt, da das Ziel der Inhibierung ein lohnendes Folgeziel für Medikamentenentwicklung darstellt, welche Allgemeingesundheit und Lebensspanne erhöhen werden. Durch Röntgenkristallographie haben wir die dimere Kristallstruktur von PorX aufgeklärt, sowohl ohne, als auch im substratgebunden Zustand, in Komplex mit pGpG. Wir beschreiben die einzelnen PorX Domänen und ihr Zusammenwirken, das katalytische Zentrum sowie die Substratbindestellen. Durch Röntgenkleinwinkelstreuung haben wir die Struktur von PorX in Lösung analysiert, als Monomer sowie Dimer. Daraufhin und durch strukturbasierte Mutationsstudien haben wir den Mechanismus der Aktivierung und Dimerisierung von PorX erklärt und durch Phosphorylierungs- analysen, Größenausschlusschromatographie (SEC) und SEC-MALLS (multi-angle-laser-light- scattering) bestätigt. Die enzymatisch aktive C-terminale Domäne von PorX gehört zur PglZ-Familie, dessen Struktur und Funktion, trotz der relativen Häufigkeit in Prokaryonten,[11] bisher unaufgeklärt war. Daher wurde in dieser Arbeit die Struktur und das katalytische Zentrum von PorX mit seinen nächsten Homologen verglichen, sowie mit bekannten PglZ Klassen der BREX Systeme. Als Ergebnis konnten wir PorX-PglZ als Phosphodiesterase der Superfamilie der alkalinen Phosphatasen (APS) identifizieren. Sowohl das aktivitätsdeterminierende katalytische Zentrum, wie auch das bisher unbeschriebene Dimerisierungsmotiv, beteiligt an enzymatischer Aktivierung, sind zwischen BREX-PglZs hoch konserviert. In PorX faltet sich dieses Motiv während des Dimerisierungsvor- ganges und ist entscheidend für die Metallkoordinierung des katalytischen Zentrums. Weiterhin konnten wir über enzymatische Analysen die Substratspezifität für PorX aufklären und zeigen dass PorX spezifisch lineare und zyklische Oligonukleotide zu Nukleotidmonophosphaten hydrolysiert. Somit ist die Funktion von PorX an der Regulation von Oligonukleotidsignaltransduktion in PG beteiligt, und durch unsere Kristallstrukturen ist es möglich, dass, durch spezifisches Design von PorX-Inhibitoren, in Zukunft die Virulenz von PG verhindert werden kann. Literaturverzeichnis 1: Veith, Paul D.; Glew, Michelle D.; Gorasia, Dhana G.; Reynolds, Eric C., Type IX secretion, 2017. 2: Hajishengallis, G.; Lamont, R. J., Beyond the red complex and into more complexity, 2012. 3: Olsen, Ingar; Yilmaz, Özlem, Modulation of inflammasome activity by Porphyromonas gingivalis in periodontitis and associated systemic diseases, 2016. 4: Huck, Olivier; You, Jian; Han, Xianxian; Cai, Bin; Panek, James; Amar, Salomon, Reduction of Articular and Systemic Inflammation by Kava-241 in a Porphyromonas gingivalis-Induced Arthritis Murine Model, 2018. 5: Jia, Lu; Han, Nannan; Du, Juan; Guo, Lijia; Luo, Zhenhua; Liu, Yi, Pathogenesis of Important Virulence Factors of Porphyromonas gingivalis via Toll-Like Receptors, 2019. 6: Gholizadeh, Pourya; Eslami, Hosein; Yousefi, Mehdi; Asgharzadeh, Mohammad; Aghazadeh, Mohammad; Kafil, Hossein Samadi, Role of oral microbiome on oral cancers, a review, 2016. 7: Reyes, Leticia; Herrera, David; Kozarov, Emil; Roldá, Silvia; Progulske-Fox, Ann, Periodontal bacterial invasion and infection, 2013. 8: Huck, O; Saadi-Thiers, K; Tenenbaum, H; Davideau, JL; Romagna, C; Laurent, Y; Cottin, Y; Roul, JG., Evaluating periodontal risk for patients at risk of or suffering from atherosclerosis, 2011. 9: Chistiakov, Dimitry A.; Orekhov, Alexander N.; Bobryshev, Yuri V., Links between atherosclerotic and periodontal disease, 2016. 10: Fiorillo, Luca; Cervino, Gabriele; Laino, Luigi; D'Amico, Cesare; Mauceri, Rodolfo; Tozum, Tolga Fikret; Gaeta, Michele; Cicciù, Marco, Porphyromonas gingivalis, Periodontal and Systemic Implications-A Systematic Review, 2019. 11: Goldfarb, T; Sberro, H; Weinstock, E; Cohen, O; Doron, Sy et al., BREX is a novel phage resistance system widespread in microbial genomes, 2015.Universitat de BarcelonaSolà Vilarrubias, MariaUniversitat de Barcelona. Facultat de Farmàcia i Ciències de l'Alimentació2023info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/203352http://hdl.handle.net/10803/689250Tesis Doctorals - Facultat - Farmàcia i Ciències de l'Alimentacióreponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaInglés(c) Schmitz, Claus-A., 2023info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/2033522026-05-27T06:46:51Z
score 15,301603

Similares en LA Referencia