title: The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.
abstract: Mannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro.
authors.affiliation: Institut de Biologie Structurale (IBS), Direction des Sciences du Vivant, Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France. christine.gaboriaud@ibs.fr
authors.forename: Christine
authors.initials: C
authors.lastname: Gaboriaud
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Gupta
authors.forename: Lydie
authors.initials: L
authors.lastname: Martin
authors.forename: Monique
authors.initials: M
authors.lastname: Lacroix
authors.forename: Laurence
authors.initials: L
authors.lastname: Serre
authors.forename: Florence
authors.initials: F
authors.lastname: Teillet
authors.forename: Gérard J
authors.initials: GJ
authors.lastname: Arlaud
authors.forename: Véronique
authors.initials: V
authors.lastname: Rossi
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Thielens
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D015923:Complement C1r / Q000737:chemistry; D003173:Complement C1s / Q000737:chemistry; D003374:Coumarins; D018360:Crystallography, X-Ray; D057075:Enzyme Assays; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes; D006801:Humans; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000737:chemistry* / Q000235:genetics; D062105:Molecular Docking Simulation; D033903:Periplasmic Proteins / Q000737:chemistry*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D003374:Coumarins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; C084067:MASP1 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases; D014357:Trypsin; D015923:Complement C1r; D003173:Complement C1s; D013917:Thrombin