Latency, thermal stability, and identification of an inhibitory compound of mirolysin, a secretory protease of the human periodontopathogen Tannerella forsythia

Krzysztof M Zak; Mark J Bostock; Irena Waligorska; Ida B Thøgersen; Jan J Enghild; Grzegorz M Popowicz; Przemyslaw Grudnik; Jan Potempa; Miroslaw Ksiazek

doi:10.1080/14756366.2021.1937619

Latency, thermal stability, and identification of an inhibitory compound of mirolysin, a secretory protease of the human periodontopathogen Tannerella forsythia

J Enzyme Inhib Med Chem. 2021 Dec;36(1):1267-1281. doi: 10.1080/14756366.2021.1937619.

Authors

Krzysztof M Zak^{1

2}, Mark J Bostock^{1

3}, Irena Waligorska⁴, Ida B Thøgersen⁵, Jan J Enghild⁵, Grzegorz M Popowicz^{1

3}, Przemyslaw Grudnik², Jan Potempa^{4

6}, Miroslaw Ksiazek^{4

6}

Affiliations

¹ Helmholtz Zentrum München, Institute of Structural Biology, Neuherberg, Germany.
² Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland.
³ Biomolecular NMR and Center for Integrated Protein Science Munich at Department Chemie, Technical University of Munich, Garching, Germany.
⁴ Department of Microbiology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland.
⁵ Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
⁶ Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY, USA.

Abstract

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (K_i = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.

Keywords: NMR-based fragment screening; Periodontitis; Tannerella forsythia; protease inhibitors; proteolysis.

MeSH terms

Bacterial Proteins / drug effects
Bacterial Proteins / metabolism
Drug Discovery
Electrophoresis, Polyacrylamide Gel
Enzyme Stability
Humans
Magnetic Resonance Spectroscopy / methods
Molecular Docking Simulation
Molecular Structure
Peptide Hydrolases / drug effects
Peptide Hydrolases / metabolism*
Periodontitis / microbiology*
Protease Inhibitors / chemistry
Protease Inhibitors / pharmacology*
Tannerella forsythia / enzymology*
Tannerella forsythia / isolation & purification
Temperature

Substances

Bacterial Proteins
Protease Inhibitors
Peptide Hydrolases

Grants and funding

This work was supported by the National Science Center, Poland, under grant numbers [UMO-2019/35/B/NZ1/03118] to M.K. and UMO-2018/31/N/NZ1/02891 to I.W.; and the Polish Ministry of Science and Higher Education under grant number [1306/MOB/IV/2015/0] (Mobility Plus to M.K.). M.J.B. is supported by the Helmholtz Zentrum München. We also acknowledge the MCB Structural Biology Core Facility (supported by the TEAM TECH CORE FACILITY/2017–4/6 grant from the Foundation for Polish Science) for valuable support. This research was supported in part by PLGrid Infrastructure. We are grateful to Dr. Sam Asami and Dr. Gerd Gemmecker (BNMRZ, München) for support with NMR experiments.