The role of glycerol-water mixtures in the stability of FKBP12-rapalog-FRB complexes.

The thermodynamic and biophysical implications of the introduction of a co-solvent during protein-ligand binding remain elusive. Using ternary complexes of 12-kDa FK506 binding protein (FKBP12), FKBP-rapamycin binding (FRB) domain of the mammalian/mechanistic target of rapamycin (mTOR) kinase, and rapamycin analogs (rapalogs) in glycerol-water mixtures, the influence of solvent composition on ligand binding dynamics was explored. The pharmaceutical potential of rapalogs and the utility of glycerol as a co-solvent in drug delivery applications were critical in deciding the system to be studied. Consolidation of existing studies on rapamycin modification was first performed to strategically design a new rapalog called T1. The results from 100-ns dual-boost Gaussian accelerated molecular dynamics simulations showed that protein stability was induced in the presence of glycerol. Reweighting of the trajectories revealed that the glycerol-rich solvent system lowers the energy barrier in the conformational space of the protein while also preserving native contacts between the ligand and the residues in the binding site. Calculated binding free energies using MM/GBSA also showed that electrostatic energy and polar contribution of solvation energy are heavily influenced by the changes in solvation. Glycerol molecules are preferentially excluded through electrostatic interactions from the solvation shell which induce complex stability as seen in existing experiments. Hence, using glycerol as a co-solvent in rapamycin delivery has a significant role in maintaining stability. In addition, compound T1 is a potential mTORC1-selective inhibitor with strong affinity for the FKBP12-FRB complex. This study aims to provide insights on the design of new rapalogs, and the applicability of glycerol as co-solvent for FKBP12-rapalog-FRB complexes.

Reparameterization of Non-Bonded Parameters for Copper Ions in Plastocyanin: An Adaptive Force Matching Study.

Molecular mechanics rely on existing experimental and theoretical inputs to confidently calculate the trajectories of molecular systems. These calculations, however, are often hindered by missing force field parameters. A notable subject of this problem is metal centers of proteins. This study parameterized, through an adaptive force matching (AFM) workflow, the copper cofactor of plastocyanin in its two oxidation states. New 12-6 Lennard-Jones (LJ) parameters and atomic partial charges were generated to complete the non-bonded description of the copper site. Our models show uniform distorted tetrahedral structures for reduced plastocyanin, Cu(I), and oxidized plastocyanin, Cu(II). These structures align with the QM/MM MD results and existing crystallography studies. TD-DFT calculations, meanwhile, showed that conformations with elongated axial Cu-SMet and shortened equatorial Cu-SCys bonds retain the experimental UV-Vis profile of blue copper (BC) proteins, thus signifying the importance of Cu-S interactions on BC proteins' unique spectroscopic properties.

pH-Dependent Conformations of an Antimicrobial Spider Venom Peptide, Cupiennin 1a, from Unbiased HREMD Simulations.

Cupiennin 1a is an antimicrobial peptide found in the venom of the spider Cupiennius salei. A highly cationic peptide, its cell lysis activity has been found to vary between neutral and charged membranes. In this study, Hamiltonian replica-exchange molecular dynamics (HREMD) was used to determine the conformational ensemble of the peptide in both charged (pH 3) and neutral (pH 11) states. The obtained free energy landscapes demonstrated the conformational diversity of the neutral peptide. At high pH, the peptide was found to adopt helix-hinge-helix and disordered structures. At pH 3, the peptide is structured with a high propensity toward α-helices. The presence of these α-helices seems to assist the peptide in recognizing membrane surfaces. These results highlight the importance of the charged residues in the stabilization of the peptide structure and the subsequent effects of pH on the peptide's conformational diversity and membrane activity. These findings may provide insights into the antimicrobial activity of Cupiennin 1a and other amphipathic linear peptides toward different cell membranes.

Identification of Conomarphin Variants in the Conus eburneus Venom and the Effect of Sequence and PTM Variations on Conomarphin Conformations.

Marine cone snails belonging to the Conidae family make use of neuroactive peptides in their venom to capture prey. Here we report the proteome profile of the venom duct of Conus eburneus, a cone snail belonging to the Tesseliconus clade. Through tandem mass spectrometry and database searching against the C. eburneus transcriptome and the ConoServer database, we identified 24 unique conopeptide sequences in the venom duct. The majority of these peptides belong to the T and M gene superfamilies and are disulfide-bonded, with cysteine frameworks V, XIV, VI/VII, and III being the most abundant. All seven of the Cys-free peptides are conomarphin variants belonging to the M superfamily that eluted out as dominant peaks in the chromatogram. These conomarphins vary not only in amino acid residues in select positions along the backbone but also have one or more post-translational modifications (PTMs) such as proline hydroxylation, C-term amidation, and γ-carboxylation of glutamic acid. Using molecular dynamics simulations, the conomarphin variants were predicted to predominantly have hairpin-like or elongated structures in acidic pH. These two structures were found to have significant differences in electrostatic properties and the inclusion of PTMs seems to complement this disparity. The presence of polar PTMs (hydroxyproline and γ-carboxyglutamic acid) also appear to stabilize hydrogen bond networks in these conformations. Furthermore, these predicted structures are pH sensitive, becoming more spherical and compact at higher pH. The subtle conformational variations observed here might play an important role in the selection and binding of the peptides to their molecular targets.

Conformational dynamics of [Formula: see text]-conotoxin PnIB in complex solvent systems.

Cone snails are slow-moving animals that secure survival by injecting to their prey a concoction of highly potent and stable neurotoxic peptides called conotoxins. These small toxins (~ 10-30 AA) interact with ion channels and their diverse structures account for various variables such as the environment and the prey of preference. This study probed the conformational space of α-conotoxin PnIB from Conus pennaceus by performing all-atom molecular dynamics simulations on the conotoxin in complex solvent systems of water and octanol. Secondary structure analyses showed a uniform conformation for the pure (C100Oc, C100W) and minute (C95Oc, C5Oc) systems. In C50Oc, however, structural changes were observed. The original helices were converted to turns and were shown to happen simultaneously with the elongation of the helix and shortening of end-to-end distance. The transitions complement the orientation of the peptide at the interface. The shift to the broken helix conformation is marked by the rearrangement of solvent molecules to a framework that favors the accumulation of water molecules at residues 6-11 of the H2 region. This promotes specific protein-solvent interactions that facilitate secondary structure transitions. As PnIB has shown favorable binding toward neuronal nicotinic acetylcholine receptors, this study may provide insights on this conotoxin's therapeutic potential. Description: Structural changes in PnIB are accompanied by a simultaneous change in solvent density.