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

International audienceMannan-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

A methodology and an instrument for the temperature-controlled optimization of crystal growth Acta Crystallographica D, 2007, vol.

A method and a device for the promotion of crystal growth by keeping the crystallization solution metastable during the growth process are described. This is achieved by controlled temperature variation of the crystallization solution using parameters determined in situ during the growth process. The technique finds application in the growth of large high-quality crystals for neutron crystallography. Thus, it has been applied to grow large crystals of several proteins of interest such as human gamma-crystallin E, PA-IIL lectin from Pseudomonas aeruginosa, yeast inorganic pyrophosphatase, urate oxidase from Aspergillus flavus and human carbonic anhydrase II

Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states in Aspergillus flavus urate oxidase

Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05–1.20 Å) and neutron diffraction data (1.9–2.5 Å) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H2O and D2O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis

A preliminary neutron diffraction study of Rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 angstrom resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm(3). The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high- resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit- cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 angstrom) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons

Interfacial assembly of proteins and peptides: recent examples studied by neutron reflection

Through reviewing a number of recent neutron reflection studies of interfacial adsorption of peptides and proteins, this paper aims to demonstrate the significance of this technique in studying interfacial biomolecular processes by illustrating the typical structural details that can be derived. The review will start with the introduction of relevant theoretical background, followed by an outline of representative biomolecular systems that have recently been studied to indicate the technical strengths of neutron reflection