The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.

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.

Antitumoral activity of allosteric inhibitors of protein kinase CK2.

INTRODUCTION: Due to its physiological role into promoting cell survival and its dysregulation in most cancer cells, protein kinase CK2 is a relevant physiopathological target for development of chemical inhibitors. We report the discovery of azonaphthalene derivatives, as a new family of highly specific CK2 inhibitors. First, we demonstrated that CK2 inhibition (IC50= 0.4 µM) was highly specific, reversible and non ATP-competitive. Small Angle X-ray Scattering experiments showed that this inhibition was due to large conformational change of CK2α upon binding of these inhibitors. We showed that several compounds of the family were cell-potent CK2 inhibitors promoting cell cycle arrest of human glioblastoma U373 cells. Finally, in vitro and in vivo assays showed that these compounds could decrease U373 cell tumor mass by 83 % emphasizing their efficacy against these apoptosis-resistant tumors. In contrast, Azonaphthalene derivatives inactive on CK2 activity showed no effect in colony formation and tumor regression assays. These findings illustrate the emergence of nonclassical CK2 inhibitors and provide exciting opportunities for the development of novel allosteric CK2 inhibitors. BACKGROUND: CK2 is an emerging therapeutic target and ATP-competitive inhibitors have been identified. CK2 is endowed with specific structural features providing alternative strategies for inhibition. RESULTS: Azonaphthalene compounds are allosteric CK2 inhibitors showing antitumor activity. CONCLUSION: CK2 may be targeted allosterically. SIGNIFICANCE: These inhibitors provide a foundation for a new paradigm for specific CK2 inhibition.

Structure and properties of the Ca(2+)-binding CUB domain, a widespread ligand-recognition unit involved in major biological functions.

CUB domains are 110-residue protein motifs exhibiting a ß-sandwich fold and mediating protein-protein interactions in various extracellular proteins. Recent X-ray structural and mutagenesis studies have led to the identification of a particular CUB domain subset, cbCUB (Ca(2+)-binding CUB domain). Unlike other CUB domains, these harbour a homologous Ca(2+)-binding site that underlies a conserved binding site mediating ionic interaction between two of the three conserved acidic Ca(2+) ligands and a basic (lysine or arginine) residue of a protein ligand, similar to the interactions mediated by the low-density lipoprotein receptor family. cbCUB-mediated protein-ligand interactions usually involve multipoint attachment through several cbCUBs, resulting in high-affinity binding through avidity, despite the low affinity of individual interactions. The aim of the present review is to summarize our current knowledge about the structure and functions of cbCUBs, which represent the majority of the known CUB repertoire and are involved in a variety of major biological functions, including immunity and development, as well as in various cancer types. Examples discussed in the present review include a wide range of soluble and membrane-associated human proteins, as well as some archaeal and invertebrate proteins. The fact that these otherwise unrelated proteins share a common Ca(2+)-dependent ligand-binding ability suggests a mechanism inherited from very primitive ancestors. The information provided in the present review should stimulate further investigations on the crucial interactions mediated by cbCUB-containing proteins.

Biochemical and structural analysis of the binding determinants of a vascular endothelial growth factor receptor peptidic antagonist.

Cyclic peptide antagonist c[YYDEGLEE]-NH(2), which disrupts the interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), represents a promising tool in the fight against cancer and age-related macular degeneration. Furthermore, coupled to a cyclen derivative, this ligand could be used as a medicinal imaging agent. Nevertheless, before generating such molecular probes, some preliminary studies need to be undertaken in order to define the more suitable positions for introduction of the cyclen macrocycle. Through an Ala-scan study on this peptide, we identified its binding motif, and an NMR study highlights its binding sites on the VEGFR-1D2 Ig-like domain. Guided by the structural relationship results deduced from the effect of the peptides on endothelial cells, new peptides were synthesized and grafted on beads. Used in a pull-down assay, these new peptides trap the VEGFRs, thus confirming that the identified amino acid positions are suitable for further derivatization.

Crystal structure of the CUB1-EGF-CUB2 domain of human MASP-1/3 and identification of its interaction sites with mannan-binding lectin and ficolins.

MASP-1 and MASP-3 are homologous proteases arising from alternative splicing of the MASP1/3 gene. They include an identical CUB(1)-EGF-CUB(2)-CCP(1)-CCP(2) module array prolonged by different serine protease domains at the C-terminal end. The x-ray structure of the CUB(1)-EGF-CUB(2) domain of human MASP-1/3, responsible for interaction of MASP-1 and -3 with their partner proteins mannan-binding lectin (MBL) and ficolins, was solved to a resolution of 2.3A(.) The structure shows a head-to-tail homodimer mainly stabilized by hydrophobic interactions between the CUB(1) module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca(2+) ion bound primarily to both EGF modules stabilizes the intra- and inter-monomer CUB(1)-EGF interfaces. Additional Ca(2+) ions are bound to each CUB(1) and CUB(2) module through six ligands contributed by Glu(49), Asp(57), Asp(102), and Ser(104) (CUB(1)) and their counterparts Glu(216), Asp(226), Asp(263), and Ser(265) (CUB(2)), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L- and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB(1) and CUB(2), located in close vicinity of their Ca(2+)-binding sites and stabilized by the Ca(2+) ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca(2+) ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL.MASP complex is proposed.

Flavonoids as RTK inhibitors and potential anticancer agents.

Tyrosine kinase receptors (RTKs) play a crucial role in the regulation of the cell division cycle. Currently more than 50 RTKs divided into several subfamilies have been described. The inhibition of these enzymes has emerged as an important research-area. Compounds able to inhibit the activity of these enzymes are expected to display antiproliferative properties. Flavonoids are representative of various small molecules acting as RTK inhibitors. These naturally occurring compounds are able to bind to the ATP-binding site of several kinases. The most plausible current hypothesis explaining the action of these substances on kinases is that the chromenone moiety of the flavonoid acts as a mimetic of the adenine moiety of ATP, the receptor co-factor. In this review, we report recent results on the activity of natural and synthetic derivatives of flavonoids as inhibitors of RTKs. Mechanistic aspects, the therapeutic usefulness, and the potential clinical use are discussed.

Assembly of C1 and the MBL- and ficolin-MASP complexes: structural insights.

The classical pathway C1 complex, and the MBL-MASP and ficolin-MASP complexes involved in activation of the lectin pathway have several features in common. Both types of complexes are assembled from two subunits: an oligomeric recognition protein (C1q, MBL, L-, H- or M-ficolin), and a protease component, which is either a tetramer (C1s-C1r-C1r-C1s) or a dimer ((MASP)(2)). Recent functional and 3-D structural investigations have revealed that C1r/C1s and the MASPs associate through a common mechanism involving their N-terminal CUB1-EGF region. In contrast, the C1s-C1r-C1r-C1s tetramer and the (MASP)(2) dimers appear to have evolved distinct strategies to associate with their partner proteins. The purpose of this article is to review these recent advances.

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55.

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

Functional characterization of complement proteases C1s/mannan-binding lectin-associated serine protease-2 (MASP-2) chimeras reveals the higher C4 recognition efficacy of the MASP-2 complement control protein modules.

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.

The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases.

Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 +/- 170 Da (MBL-I) and 304,899 +/- 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 +/- 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable K(D) values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.