Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani

LC MS/MS analysis of identified interactors of mASAL from R.solani Matched peptides highlighted in yellow. Green colour indicated probable sites of mutation and modification. (DOCX 2225 kb

Capturing a Crucial ‘Disorder-to-Order Transition’ at the Heart of the Coronavirus Molecular Pathology—Triggered by Highly Persistent, Interchangeable Salt-Bridges

The COVID-19 origin debate has greatly been influenced by genome comparison studies of late, revealing the emergence of the Furin-like cleavage site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCSSpike) containing its 681PRRAR685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a ‘disorder-to-order transition’ of the FLCSSpike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike–Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCSSpike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seem to be the other key characteristic features of the Spike–Furin binding in SARS-CoV-2, allowing the system to breathe—a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike–Furin binding events, generally, in the coronavirus lineage, seems to have preference for ionic bond formation, even with a lesser number of cationic residues at their potentially polybasic FLCSSpike patches. The interaction energies are suggestive of characteristic metastabilities attributed to Spike–Furin interactions, generally to the coronavirus lineage, which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. The current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution, but also add substantially to the existing theories of proteolytic cleavages

A Co(II)-Hydrazone Schiff Base Single Ion Magnet Exhibiting Field Induced Slow Relaxation Dynamics

An octahedral Co(II) complex with N′-(2-hydroxybenzylidene)acetohydrazide Schiff base ligand [HL] forms a 3D supramolecular assembly supported by non-coordinating ClO4− ions and H2O molecules. Individual spin centres are non-interacting and give rise to significant spin-orbit coupling, resulting in field induced slow magnetisation relaxation; which is characteristic of Single Ion Magnet (SIM) behaviour

Additional file 2: Table S1. of Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani

Excel spreadsheet showing total peptides fragments used to identify proteins. (XLSX 43 kb

Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

BackgroundAllium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL.Methodology/principal findingsBy introduction of 5 site-specific mutations (-DNSNN-), a β turn was incorporated between the 11(th) and 12(th) β strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL). mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and Alternaria brassicicola in a disc diffusion assay. A propidium iodide uptake assay suggested that the inhibitory activity of mASAL might be associated with the alteration of the membrane permeability of the fungus. Furthermore, a ligand blot assay of the membrane subproteome of R. solani with mASAL detected a glycoprotein receptor having interaction with mASAL.Conclusions/significanceConversion of ASAL into a stable monomer resulted in antifungal activity. From an evolutionary aspect, these data implied that variable quaternary organization of lectins might be the outcome of defense-related adaptations to diverse situations in plants. Incorporation of mASAL into agronomically-important crops could be an alternative method to protect them from dramatic yield losses from pathogenic fungi in an effective manner

Additional file 1: Figure S1. of Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani

Nuclei counts in hyphae of control and mASAL treated fungal hyphae. Hyphae were harvested and stained with DAPI. The number of nuclei between the hyphal tip and the first two septum was counted manually. For the control type and mASAL treated sample, the graph shows the average number ± S.D. of nuclei counted in at three different time points (*, P < 0.05). (DOCX 636 kb

Biological Safety Assessment of Mutant Variant of <i>Allium sativum</i> Leaf Agglutinin (mASAL), a Novel Antifungal Protein for Future Transgenic Application

Genetic engineering has established itself to be an important tool for crop improvement. Despite the success, there is always a risk of food allergy induced by alien gene products. The present study assessed the biosafety of mutant <i>Allium sativum</i> leaf agglutinin (mASAL), a potent antifungal protein generated by site directed mutagenesis of <i>Allium sativum</i> leaf agglutinin (ASAL). mASAL was cloned in pET28a+ and expressed in <i>E. coli</i>, and the safety assessment was carried out according to the FAO/WHO guideline (2001). Bioinformatics analysis, pepsin digestion, and thermal stability assay showed the protein to be nonallergenic. Targeted sera screening revealed no significant IgE affinity of mASAL. Furthermore, mASAL sensitized Balb/c mice showed normal histopathology of lung and gut tissue. All results indicated the least possibility of mASAL being an allergen. Thus, mASAL appears to be a promising antifungal candidate protein suitable for agronomical biotechnology