Crystal Structure of Mistletoe Lectin I (ML-I) from Viscum album in Complex with 4-N-Furfurylcytosine at 2.85 Å Resolution

ackground: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification.Methods: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified by in-silico docking.Results: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1.Conclusion: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain, and related RIPs

SAXS and other spectroscopic analysis of 12S cruciferin isolated from the seeds of Brassica nigra

Oilseeds of the plant family Brassicaceae are important for providing both lipid and protein contents to human nutrition. Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (∼190 kDa) cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30% saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with cruciferin from Arabidopsis thaliana. The 50–55 kDa monomeric cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28–32 kDa and β-polypeptides of 17–20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7 and 8.0 (α-polypeptides) and 5.5–8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8 Å). Small-angle x-ray scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution. SAXS showed that the radius of gyration is 4.24 ± 0.25 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra cruciferin was compared with 11S globulins (PDB ID: 3KGL) of B. napus which further confirmed a highly similar molecular weight and globular shape indicating a conserved trimerization of B. nigra cruciferin. The comparison of the scattering patterns of both proteins showed a minimized χ2-value of 1.337 confirming a similar molecular structure. This is the first report describing the purification and characterization of a cruciferin protein from seeds of B. nigra

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl b-DN, N0,N00-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (b/a)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 mg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 mg/well Similarly, insect toxicity bioassays of GgChi (50 mg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase

Eruca sativa seed napin structural insights and thorough functional characterization

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC–MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an R$_g$ of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm$^3$, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ$^2$-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl β-d-N,N′,N′′-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (β/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 μg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 μg/well Similarly, insect toxicity bioassays of GgChi (50 μg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase. Keywords: Gladiolus grandiflorus, Endochitinase, TIM barrel, Antibacterial, Antifunga