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...
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| 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 |
| Sumario: | [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. |
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