Sequence analysis and crystal structure of a glycosylated protease from Euphorbia resinifera latex for its proteolytic activity aspect.

The investigation of a plant glycosylated serine protease (EuRP-61) isolated from Euphorbia resinifera latex for potential antiplatelet and anticoagulation activities has been previously reported. In the present study, the protein sequence and native crystal structure of EuRP-61 were characterized. The structure was identified using single-wavelength anomalous diffraction with a refinement resolution of 1.7 Å (PDB ID: 7EOX). The main structural components of EuRP-61 were composed of three domains: catalytic, protease-associated (PA), and fibronectin type III (Fn3)-like domains. The crystal structure revealed that some loops in the PA and catalytic domains of EuRP-61 were different from the other subtilisin-like proteases (cucumisin and SBT3). These different loops might be involved in the general monomer formation of EuRP-61, substrate specificity, and maintenance of the catalytic domain. The Fn3-like domain may provide flexibility to the enzyme to bind with various substrates and cell receptors. Additionally, the active site of EuRP-61 consisted of the catalytic triad of Ser434, His106, and Asp32, similar to other serine proteases. The present study provides additional information and insight into the protease and antithrombotic activities of EuRP-61, which could contribute to further development of this enzyme for biomedical treatment.

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding.

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded ß-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

Identification and characterization of a protease (EuRP-61) from Euphorbia resinifera latex.

A serine protease designated as EuRP-61 was purified from Euphorbia resinifera latex. The N-terminal sequence of 15 amino acids of EuRP-61 supported the conclusion that the enzyme was a serine protease because its amino acid sequence had homology (between 50 and 70% identities) with the subtilisin-like proteases of other plants. EuRP-61 had a molecular weight estimated at 61 kDa analyzed by MALDI-TOF MS. The enzyme could cleave human fibrinogen with optimal conditions at pH 5.0 and 45 °C. The enzyme had a broad range of pH stability from 1 to 14 and tolerance to denaturation up to a temperature of approximately 65-66 °C. EuRP-61 hydrolyzed fibrinogen with a Michaelis constant (Km) of 4.95 ±â€¯0.1 µM; a maximal velocity (Vmax) of 578.1 ±â€¯11.81 ng min-1; and a catalytic efficiency (Vmax/Km) of 116.8 ±â€¯1 ng µM-1 min-1. EuRP-61was crystallized under the condition of sodium iodide (0.2 M), Bis-Tris propane (0.1 M, pH 8.5) and PEG3350 (20%) by the sitting-drop method. The crystal belonged to space group P212121, with unit cell dimension a = 109.91, b = 67.38 and c = 199.45 Šand diffracted X-ray to 2.53 Šresolution. The crystal structure of EuRP-61 will be explored further by special phase solving techniques.