Synergy of protease-binding sites within the ecotin homodimer is crucial for inhibition of MASP enzymes and for blocking lectin pathway activation.
Binding Sites
Complement Pathway, Mannose-Binding Lectin
Escherichia coli
/ genetics
Escherichia coli Proteins
/ chemistry
Lectins
/ genetics
Mannose-Binding Lectin
/ metabolism
Mannose-Binding Protein-Associated Serine Proteases
/ chemistry
Peptide Hydrolases
/ metabolism
Periplasmic Proteins
/ chemistry
Protein Subunits
X-ray crystallography
complement system
ecotin
lectin pathway
mannose-binding lectin-associated serine protease
virulence factor
Journal
The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R
Informations de publication
Date de publication:
06 2022
06 2022
Historique:
received:
06
10
2021
revised:
19
04
2022
accepted:
22
04
2022
pubmed:
29
4
2022
medline:
30
6
2022
entrez:
28
4
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35483450
pii: S0021-9258(22)00425-2
doi: 10.1016/j.jbc.2022.101985
pmc: PMC9136129
pii:
doi:
Substances chimiques
Eco protein, E coli
0
Escherichia coli Proteins
0
Lectins
0
Mannose-Binding Lectin
0
Periplasmic Proteins
0
Protein Subunits
0
Peptide Hydrolases
EC 3.4.-
MASP1 protein, human
EC 3.4.21.-
MASP2 protein, human
EC 3.4.21.-
Mannose-Binding Protein-Associated Serine Proteases
EC 3.4.21.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
101985Commentaires et corrections
Type : ErratumIn
Informations de copyright
Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
Références
J Mol Biol. 1998 Jun 19;279(4):945-57
pubmed: 9642073
PLoS Pathog. 2019 Dec 20;15(12):e1008232
pubmed: 31860690
Protein Sci. 1995 Feb;4(2):141-8
pubmed: 7757004
Biochem J. 1953 May;54(2):347-52
pubmed: 13058883
J Mol Biol. 2001 May 18;308(5):975-91
pubmed: 11352586
Protein Sci. 2018 Jan;27(1):293-315
pubmed: 29067766
J Mol Biol. 2004 Oct 1;342(5):1533-46
pubmed: 15364579
J Immunol. 2009 Sep 1;183(5):3409-16
pubmed: 19667088
J Biol Chem. 1997 Nov 28;272(48):29987-90
pubmed: 9374470
Immunobiology. 2018 Dec;223(12):744-749
pubmed: 30033110
PLoS One. 2013 Jul 04;8(7):e67962
pubmed: 23861840
Sci Rep. 2016 Aug 18;6:31877
pubmed: 27535802
Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92
pubmed: 3881765
Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
pubmed: 21460441
J Biol Chem. 2002 Jul 19;277(29):26623-31
pubmed: 11959867
Proc Natl Acad Sci U S A. 2012 Jun 26;109(26):10498-503
pubmed: 22691502
Front Immunol. 2018 Jan 29;9:63
pubmed: 29434593
FEBS Lett. 1995 May 29;365(2-3):159-63
pubmed: 7781771
Acta Biochim Pol. 1999;46(3):531-65
pubmed: 10698264
EMBO J. 1994 Apr 1;13(7):1502-7
pubmed: 8156987
FEBS Lett. 1996 May 6;385(3):165-70
pubmed: 8647243
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1410-5
pubmed: 11171964
J Immunol. 2010 Oct 1;185(7):4169-78
pubmed: 20817870
FEBS Lett. 1994 Mar 28;342(1):57-60
pubmed: 8143850
Biochemistry. 2001 Aug 28;40(34):10038-46
pubmed: 11513582
Int Arch Allergy Appl Immunol. 1977;54(5):428-33
pubmed: 885627
J Biol Chem. 1994 Aug 26;269(34):21915-8
pubmed: 8063835
Biochemistry. 1994 Apr 5;33(13):3949-58
pubmed: 8142399
Cell Mol Life Sci. 2003 Nov;60(11):2427-44
pubmed: 14625687
J Mol Biol. 2000 Jun 16;299(4):993-1003
pubmed: 10843853
J Immunol. 2009 Jul 15;183(2):1207-14
pubmed: 19564340
Clin Exp Immunol. 1999 Jun;116(3):401-9
pubmed: 10361226
Immunogenetics. 2006 Sep;58(9):701-13
pubmed: 16896831
Biochem J. 2004 Apr 1;379(Pt 1):107-18
pubmed: 14705961
J Biol Chem. 2011 Jul 8;286(27):24015-22
pubmed: 21531711
J Immunol Methods. 2005 Jan;296(1-2):187-98
pubmed: 15680163
J Med Microbiol. 2010 Sep;59(Pt 9):1089-1100
pubmed: 20522626
Expert Rev Anti Infect Ther. 2013 Mar;11(3):297-308
pubmed: 23458769
J Immunol. 2005 Apr 15;174(8):4998-5006
pubmed: 15814730
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
pubmed: 20383002
Biochemistry. 1982 May 11;21(10):2274-84
pubmed: 7046785
Biochim Biophys Acta. 2008 Sep;1784(9):1294-300
pubmed: 18456010
Microb Pathog. 2014 Feb-Mar;67-68:55-8
pubmed: 24462575
J Clin Microbiol. 2010 Mar;48(3):908-14
pubmed: 20042630
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
pubmed: 20124702
Mol Immunol. 2009 Sep;46(14):2745-52
pubmed: 19477526
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
pubmed: 20124692
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Sep 1;64(Pt 9):781-4
pubmed: 18765903
J Immunol. 2006 Oct 15;177(8):5471-9
pubmed: 17015733
Biochemistry. 1997 May 6;36(18):5381-92
pubmed: 9154920
Mol Microbiol. 2004 Feb;51(3):659-74
pubmed: 14731270
Mol Immunol. 2014 Oct;61(2):69-78
pubmed: 24935208
J Immunol. 2003 Feb 1;170(3):1374-82
pubmed: 12538697
Annu Rev Biochem. 1980;49:593-626
pubmed: 6996568
Front Immunol. 2015 Jan 30;6:17
pubmed: 25688244
Immunobiology. 2007;212(4-5):267-77
pubmed: 17544812
J Biol Chem. 2012 Jun 8;287(24):20290-300
pubmed: 22511776
Biochem Biophys Res Commun. 1967 Apr 20;27(2):157-62
pubmed: 6035483
J Biol Chem. 1983 Sep 25;258(18):11032-8
pubmed: 6411724
J Immunol. 2016 Jan 15;196(2):857-65
pubmed: 26673137
Cell Microbiol. 2009 Jan;11(1):106-20
pubmed: 19016791