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.

Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum

“…We could not detect any inhibition of C1r, while ecotin inhibited C1s with a 25.15±4.45 μM K I value representing a very weak inhibition. These findings are in agreement with previous SPR-based results of Gaboriaud et al [23] who immobilized ecotin on the chip and by injecting up to 2 μM C1s or C1r catalytic fragments, did not detect any interaction.…”

“…Ecotin orthologs are potent inhibitors of MASP-2 with K I values in the 10 −8 -10 −9 M range ( Fig 1, Table 1). E. coli ecotin inhibits MASP-2 with a K I of 11 nM, which is close to the SPRbased K D value of 24.7 nM reported by Gaboriaud et al [23]. Importantly, most ecotin orthologs are as good, or even better MASP-2 inhibitors than SGMI-2, a monospecific, high-affinity inhibitor of the enzyme we developed previously via directed evolution [21].…”

“…It is worth noting that, by using SPR, Gaboriaud et al measured a much weaker, 600 nM affinity between E. coli ecotin and MASP-3 [23]. An important difference between the two experiments is that they used a single MASP-3 SP domain construct, which lacked even the SP-structure stabilizing activation peptide.…”

Complement System Part I – Molecular Mechanisms of Activation and Regulation

“…Inhibition of the LP is influenced by MASP-3, MAp44, and MAp19 proteins, which share high sequence homology with MASP-1 and -2 and have similar binding affinity to MBL and ficolins ( 70 , 86 ). These proteins may compete with MASP-1 and -2, but are unable to cleave MASP, C2, and C4 preventing further activation of the LP cascade (Figure 4 D).…”

Multiple roles of complement MASP-1 at the interface of innate immune response and coagulation

“…The structure of zymogen MASP-3 also shows that the CCP1-CCP2 and the CCP2-SP interfaces have some flexibility. Interestingly, while mutant proenzyme and inhibited active MASP-3 (Gaboriaud et al, 2013) fragments have been crystallized successfully, the wild-type enzyme by itself has so far resisted crystallization.…”