Synergy of protease-binding sites within the ecotin homodimer is crucial for inhibition of MASP enzymes and for blocking lectin pathway activation

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1...

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Bibliographic Details
Published in:The Journal of biological chemistry 2022-06, Vol.298 (6), p.101985-101985, Article 101985
Main Authors: Nagy, Zoltán Attila, Héja, Dávid, Bencze, Dániel, Kiss, Bence, Boros, Eszter, Szakács, Dávid, Fodor, Krisztián, Wilmanns, Matthias, Kocsis, Andrea, Dobó, József, Gál, Péter, Harmat, Veronika, Pál, Gábor
Format: Article
Language:English
Subjects:
Binding Sites
Complement Pathway, Mannose-Binding Lectin
complement system
ecotin
Escherichia coli - genetics
Escherichia coli - metabolism
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - metabolism
lectin pathway
Lectins - genetics
Lectins - metabolism
Mannose-Binding Lectin - metabolism
mannose-binding lectin-associated serine protease
Mannose-Binding Protein-Associated Serine Proteases - chemistry
Mannose-Binding Protein-Associated Serine Proteases - metabolism
Peptide Hydrolases - metabolism
Periplasmic Proteins - chemistry
Periplasmic Proteins - metabolism
Protein Subunits
virulence factor
X-ray crystallography
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Summary:Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway–inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.
ISSN:0021-9258
1083-351X
DOI:10.1016/j.jbc.2022.101985