Tóth, Zoé Sára and Leveles, Ibolya and Nyíri, Kinga and Nagy, Gergely and Harmat, Veronika and Jaroentomeechai, Thapakorn and Ozohanics, Olivér and Miller, Rebecca L. and Ballesteros Álvarez, Marina and Vértessy, Beáta (Grolmuszné) and Benedek, András (2024) The homodimerization domain of the Stl repressor is crucial for efficient inhibition of mycobacterial dUTPase. SCIENTIFIC REPORTS, 14 (1). No.-27171. ISSN 2045-2322
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Abstract
The dUTPase is a key DNA repair enzyme in Mycobacterium tuberculosis, and it may serve as a novel promising anti-tuberculosis target. Stl repressor from Staphylococcus aureus was shown to bind to and inhibit dUTPases from various sources, and its expression in mycobacterial cells interfered with cell growth. To fine-tune and optimize Stl-induced inhibition of mycobacterial dUTPase, we aimed to decipher the molecular details of this interaction. Structural background of the complex between dUTPase and a truncated Stl lacking the repressor C-terminal homodimerization domain has been described, however, the effects of this truncation of Stl on enzyme binding and inhibition are still not known. Using several independent biophysical, structural and enzyme kinetic methods, here we show that lack of the repressor homodimerization domain strongly perturbs both enzyme binding and inhibition. We also investigated the role of a mycobacteria-specific loop in the Stl-interaction. Our results show that removal of this loop leads to a ten-fold increase in the apparent inhibition constant of Stl. We present a high-resolution three-dimensional structure of mycobacterial dUTPase lacking the genus-specific loop for structural insight. Our present data suggest that potent inhibition of mycobacterial dUTPase by Stl requires the wild-type full-length protein context.
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| Additional Information: | Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary Department of Applied Biotechnology and Food Science, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary Doctoral School of Biology, Institute of Biology, ELTE Eötvös Loránd University, Budapest, Hungary Laboratory of Structural Chemistry and Biology, Institute of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary HUN-REN-ELTE Protein Modelling Research Group, Hungarian Research Network, Budapest, Hungary Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, DK-2200, Denmark Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary Export Date: 25 November 2024 Correspondence Address: Tóth, Z.S.; Institute of Molecular Life Sciences, Hungary; email: toth.zoe@ttk.hu Correspondence Address: Vértessy, B.G.; Institute of Molecular Life Sciences, Hungary; email: vertessy.beata@ttk.hu Correspondence Address: Benedek, A.; Institute of Molecular Life Sciences, Hungary; email: benedek.andras@ttk.hu Chemicals/CAS: inorganic pyrophosphatase, 9024-82-2, 9033-44-7; Bacterial Proteins; dUTP pyrophosphatase; Pyrophosphatases; Repressor Proteins Funding details: Budapesti Műszaki és Gazdaságtudományi Egyetem, BME Funding details: Innovációs és Technológiai Minisztérium Funding details: European Commission, EC Funding details: European Regional Development Fund, ERDF, 2018 − 1.2.1-NKP-2018-00005 Funding details: European Regional Development Fund, ERDF Funding details: Carlsbergfondet, CF20-0412 Funding details: Carlsbergfondet Funding details: Nemzeti Kutatási Fejlesztési és Innovációs Hivatal, NKFIH, K146890, K135231, PD134324, 2022 − 1.2.2-TÉT-IPARI-UZ-2022-00003, FK137867, TKP2021-EGA-02 Funding details: Nemzeti Kutatási Fejlesztési és Innovációs Hivatal, NKFIH Funding details: European Molecular Biology Organization, EMBO, ALTF 336–2021 Funding details: European Molecular Biology Organization, EMBO Funding details: Magyar Tudományos Akadémia, MTA, VEKOP-2.3.2-16-2017-00014, VEKOP-2.3.3-15-2017-00018 Funding details: Magyar Tudományos Akadémia, MTA Funding details: Novo Nordisk Fonden, NNF, NNF22OC0073736 Funding details: Novo Nordisk Fonden, NNF Funding text 1: This work was supported by the National Research, Development and Innovation Office of Hungary (K135231, K146890, FK137867, 2018\\u2009\\u2212\\u20091.2.1-NKP-2018-00005, 2022\\u2009\\u2212\\u20091.2.2-T\\u00C9T-IPARI-UZ-2022-00003 to B.G.V., PD134324 to K.N.) and the TKP2021-EGA-02 grant, implemented with the support provided by the Ministry for Innovation and Technology of Hungary from the National Research, Development and Innovation Office. K.N. was also supported by the Parents Back to Science program of the Budapest University of Technology and Economics. G.N.N. was supported by the J\\u00E1nos Bolyai Research Scholarship of the Hungarian Academy of Sciences. The X-ray crystallographic study was also supported within projects No. VEKOP-2.3.2-16-2017-00014 and VEKOP-2.3.3-15-2017-00018 (to V.H), of the European Union and the Government of Hungary, co-financed by the European Regional Development Fund; as well as project no. 2018\\u2009\\u2212\\u20091.2.1-NKP-2018-00005 of the National Research Development and Innovation Fund of Hungary, financed under the 2018\\u2009\\u2212\\u20091.2.1-NKP funding scheme. This work was also supported by the European Molecular Biology Organization (EMBO) postdoctoral fellowship ALTF 336\\u20132021 (to T.J.). The Novo Nordisk Foundation grant NNF22OC0073736 (to R.L.M.) and the Carlsberg Foundation CF20-0412 (to R.L.M.). |
| Subjects: | Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia Q Science / természettudomány > QR Microbiology / mikrobiológia R Medicine / orvostudomány > RM Therapeutics. Pharmacology / terápia, gyógyszertan |
| SWORD Depositor: | MTMT SWORD |
| Depositing User: | MTMT SWORD |
| Date Deposited: | 04 Sep 2025 08:15 |
| Last Modified: | 04 Sep 2025 08:15 |
| URI: | https://real.mtak.hu/id/eprint/223444 |
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