Identification of a phage display-derived peptide interacting with the N-terminal region of Factor VII activating protease (FSAP) enables characterization of zymogen activation.

Increased Factor VII activating protease (FSAP) activity has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to a single nucleotide polymorphism. The activation of FSAP zymogen in plasma is mediated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear if this activation mechanism is specific and amenable to manipulation. Using a phage display approach we have identified a peptide, NNKC9/41, that activates pro-FSAP in plasma. Other commonly found zymogens in the plasma were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. Blocking the contact pathway of coagulation did not influence pro-FSAP activation by the peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41 and this was reversed by MA-FSAP-38C7 demonstrating the utility of this peptide. Identification of this peptide, and the corresponding interaction site, provides proof of principle that it is possible to activate a single protease zymogen in blood in a specific manner. Peptide NNKC/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP in more detail, elucidate its biological role.

Pharmacologically Targeting the Fibroblast Growth Factor 14 Interaction Site on the Voltage-Gated Na+ Channel 1.6 Enables Isoform-Selective Modulation

Voltage-gated Na+ (Nav) channels are the primary molecular determinant of the action potential. Among the nine isoforms of the Nav channel α subunit that have been described (Nav1.1-Nav1.9), Nav1.1, Nav1.2, and Nav1.6 are the primary isoforms expressed in the central nervous system (CNS). Crucially, these three CNS Nav channel isoforms display differential expression across neuronal cell types and diverge with respect to their subcellular distributions. Considering these differences in terms of their localization, the CNS Nav channel isoforms could represent promising targets for the development of targeted neuromodulators. However, current therapeutics that target Nav channels lack selectivity, which results in deleterious side effects due to modulation of off-target Nav channel isoforms. Among the structural components of the Nav channel α subunit that could be pharmacologically targeted to achieve isoform selectivity, the C-terminal domains (CTD) of Nav channels represent promising candidates on account of displaying appreciable amino acid sequence divergence that enables functionally unique protein–protein interactions (PPIs) with Nav channel auxiliary proteins. In medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a critical brain region of the mesocorticolimbic circuit, the PPI between the CTD of the Nav1.6 channel and its auxiliary protein fibroblast growth factor 14 (FGF14) is central to the generation of electrical outputs, underscoring its potential value as a site for targeted neuromodulation. Focusing on this PPI, we previously developed a peptidomimetic derived from residues of FGF14 that have an interaction site on the CTD of the Nav1.6 channel. In this work, we show that whereas the compound displays dose-dependent effects on the activity of Nav1.6 channels in heterologous cells, the compound does not affect Nav1.1 or Nav1.2 channels at comparable concentrations. In addition, we show that the compound correspondingly modulates the action potential discharge and the transient Na+ of MSNs of the NAc. Overall, these results demonstrate that pharmacologically targeting the FGF14 interaction site on the CTD of the Nav1.6 channel is a strategy to achieve isoform-selective modulation, and, more broadly, that sites on the CTDs of Nav channels interacted with by auxiliary proteins could represent candidates for the development of targeted therapeutics.

Systematic simulation of the interactions of Pleckstrin homology domains with membranes

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphates (PIPs). Here we report the systematic simulation of the interactions of 100 mammalian PH domains with PIP containing model membranes. Comparison with crystal structures of PH domains bound to PIP analogues demonstrates that our method correctly identifies interactions at known canonical and non-canonical sites, while revealing additional functionally important sites for interaction not observed in the crystal structure, such as for P-Rex1 and Akt1. At the family level, we find that the β1 and β2 strands and their connecting loop constitute the primary PIP interaction site for the majority of PH domains, but we highlight interesting exceptional cases. Simultaneous interaction with multiple PIPs and clustering of PIPs induced by PH domain binding are also observed. Our findings support a general paradigm for PH domain membrane association involving multivalent interactions with anionic lipids.

Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human–Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

Accurate Absorption Energy Calculations in Solution Using the Reference Interaction Site Model Self-Consistent Field Including the Constrained Spatial Electron Density Distribution

The solvation effect is an important factor determining the properties of molecules in solution. The reference interaction site model (RISM) is a powerful method to treat the solvation effect with pair-correlation functions, such as a radial distribution function. This study developed a hybrid method between quantum mechanics and RISM using the spatial electron density distributions on each atomic site (RISM-SCF-cSED). Sophisticated quantum mechanical approaches can be used to consider the solvation effect because the computational cost of RISM-SCF-cSED is reasonable. In this study, the absorption energies of 5-(dimethylamino)-2,4-pentadienal in various solutions were calculated using RISM-SCF-cSED. The experimental data were well reproduced with an average errors of ∼0.06 eV, using multi-reference perturbation theory.

Computational View of β-Asarone–Human Serum Albumin Interaction

β-Asarone (BAS), a bioactive phytochemical from the medicinal herb, Acorus calamus Linn., has shown many pharmacological activities. Computational docking studies unveiled the interaction site of BAS on the human plasma carrier, albumin. The primary binding arrangement of BAS was placed at Sudlow's Site I of HSA, which is pinpointed in subdomain IIA of albumin. Hydrophobic and van der Waals forces together with hydrogen bonds appear to secure the BAS-albumin complex. The BAS at Site I was surrounded by more hydrophobic and polar residues than those seen at Site II, as evidenced by LigPlot+. Therefore, the interaction between BAS and albumin at Site I seems to be comparatively more stable owing to more vital interactions.