Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 µM to >7.2 µM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Structure of POPC Lipid Bilayers in OPLS3e Force Field.

It is crucial for molecular dynamics simulations of biomembranes that the force field parameters give a realistic model of the membrane behavior. In this study, we examined the OPLS3e force field for the carbon-hydrogen order parameters SCH of POPC (1-palmitoyl-2-oleoylphosphatidylcholine) lipid bilayers at varying hydration conditions and ion concentrations. The results show that OPLS3e behaves similarly to the CHARMM36 force field and relatively accurately follows the experimentally measured SCH for the lipid headgroup, the glycerol backbone, and the acyl tails. Thus, OPLS3e is a good choice for POPC bilayer simulations under many biologically relevant conditions. The exception are systems with an abundancy of ions, as similarly to most other force fields OPLS3e strongly overestimates the membrane-binding of cations, especially Ca2+. This leads to undesirable positive charge of the membrane surface and drastically lowers the concentration of Ca2+ in the surrounding solvent, which might cause issues in systems sensitive to correct charge distribution profiles across the membrane.

RNA-protein complexes and force field polarizability.

Molecular dynamic (MD) simulations offer a way to study biomolecular interactions and their dynamics at the atomistic level. There are only a few studies of RNA-protein complexes in MD simulations, and here we wanted to study how force fields differ when simulating RNA-protein complexes: 1) argonaute 2 with bound guide RNA and a target RNA, 2) CasPhi-2 bound to CRISPR RNA and 3) Retinoic acid-inducible gene I C268F variant in complex with double-stranded RNA. We tested three non-polarizable force fields: Amber protein force fields ff14SB and ff19SB with RNA force field OL3, and the all-atom OPLS4 force field. Due to the highly charged and polar nature of RNA, we also tested the polarizable AMOEBA force field and the ff19SB and OL3 force fields with a polarizable water model O3P. Our results show that the non-polarizable force fields lead to compact and stable complexes. The polarizability in the force field or in the water model allows significantly more movement from the complex, but in some cases, this results in the disintegration of the complex structure, especially if the protein contains longer loop regions. Thus, one should be cautious when running long-scale simulations with polarizability. As a conclusion, all the tested force fields can be used to simulate RNA-protein complexes and the choice of the optimal force field depends on the studied system and research question.