Insights into the Binding Mode of Lipid A to the Anti-lipopolysaccharide Factor ALFPm3 from Penaeus monodon: An In Silico Study through MD Simulations.

The globally expanding threat of antibiotic resistance calls for the development of new strategies for abating Gram-negative bacterial infections. The use of extracorporeal blood cleansing devices with affinity sorbents to selectively capture bacterial lipopolysaccharide (LPS), which is the major constituent of Gram-negative bacterial outer membranes and the responsible agent for eliciting an exacerbated innate immune response in the host during infection, has received outstanding interest. For that purpose, molecules that bind tightly to LPS are required to functionalize the affinity sorbents. Particularly, anti-LPS factors (ALFs) are promising LPS-sequestrating molecules. Hence, in this work, molecular dynamics (MD) simulations are used to investigate the interaction mechanism and binding pose of the ALF isoform 3 from Penaeus monodon (ALFPm3), which is referred to as "AL3" for the sake of simplicity, and lipid A (LA, the component of LPS that represents its endotoxic principle). We concluded that hydrophobic interactions are responsible for AL3-LA binding and that LA binds to AL3 within the protein cavity, where it buries its aliphatic tails, whereas the negatively charged phosphate groups are exposed to the medium. AL3 residues that are key for its interaction with LA were identified, and their conservation in other ALFs (specifically Lys39 and Tyr49) was also analyzed. Additionally, based on the MD-derived results, we provide a picture of the possible AL3-LA interaction mechanism. Finally, an in vitro validation of the in silico predictions was performed. Overall, the insights gained from this work can guide the design of novel therapeutics for treating sepsis, since they may be significantly valuable for designing LPS-sequestrating molecules that could functionalize affinity sorbents to be used for extracorporeal blood detoxification.

On the effects of induced polarizability at the water-graphene interface via classical charge-on-spring models.

Molecular models of the water-graphene interaction are essential to describe graphene in condensed phases. Different challenges are associated with the generation of these models, in particular π-π and dispersion interactions; thus quantum and classical models have been developed and due to the numerical efficiency of the latter, they have been extensively employed. In this work, we have systematically studied, via molecular dynamics, two polarizable graphene models, denominated CCCP and CCCPD, employing the charge-on-spring model of the GROMOS forcefield, both being compatible with the polarizable water models COS/G2 and COS/D2, respectively. These models were compared with non-polarizable graphene and SPC water models. We focused the study on the water-graphene interface in two distinct systems and under the influence of an electric field: one composed of graphene immersed in water and the other composed of graphene with a water droplet above it. In the former, the orientation of water close to the graphene layer is affected by polarizable graphene in comparison to non-polarizable graphene. This effect is emphasised when an electric field is applied. In the latter, carbon polarizability reduced water contact angles, but graphene retained its hydrophobicity and the computed angles are within the experimental data. Given the significant extra computational cost, the use of polarizable models instead of the traditional fixed-charged approach for the graphene-water interaction may be justified when polarizability effects are relevant, for example, when applying relatively strong fields or in very anisotropic systems, such as the vacuum-bulk interface, as these models are more responsive to such conditions.

Exploring the structure and dynamics of proteins in soil organic matter.

Alongside inorganic materials, water, and air, soil organic matter (SOM) is one of the major components of soil and has tremendous influence on the environment given its vital role in the carbon cycle. Many soil dwelling organisms like plants, fungi and bacteria excrete proteins, whose interaction with SOM is poorly understood on an atomistic level. In this study, molecular dynamics simulations were used to investigate selected proteins in soil models of different complexity from simple co-solvent molecules to Leonardite humic acids (LHA). We analyzed the proteins in terms of their structural stability, the nature and strength of the interactions with their surroundings, as well as their aggregation behavior. Upon insertion of proteins in complex SOM models, their structural stability decreased, although no unfolding or disruption of secondary structure was observed. The interactions of proteins and SOM were primarily governed by electrostatic forces, often in form of hydrogen bonds. However, also weaker van der Waals forces made a significant contribution to the total interaction energies. Moreover, we showed that even though the molecular structure and size of SOM molecules varied, the functional groups of SOM ordered around the protein in a similar pattern. Finally, the number of aggregates formed by proteins and SOM molecules was shown to be primarily proportional to the size of the latter. Strikingly, for varying protein net charges no changes in the formation of aggregates with the strongly negatively charged LHA were observed.

Vienna soil organic matter modeler 2 (VSOMM2).

Soil Organic Matter (SOM) plays an important role in several biogeochemical processes by directly affecting the microbial activity, soil aggregation, plant growth and carbon storage. Despite of its importance, our understanding of its composition and structure is still incomplete. Several experiments using elemental analysis, nuclear magnetic resonance (NMR) and mass spectrometry (MS) shed light on the structure of organic matter. In this context, the Vienna Soil-Organic-Matter Modeler (https://somm.boku.ac.at/) is a website that generates condensed phase computer models of Soil-Organic-Matter (SOM). Most of the data comes from standardized samples by the International Humic Substances Association (IHSS), which uses a specific methodology to extract organic compounds from soil, called humic substances. We have improved the modeler by increasing the pool of elemental units that compose our SOM molecules called building blocks, and also by implementing a genetic algorithm that increases the chemical and geometric diversity of the models. This allowed us to create models using the IHSS data as well as different types of soil. The webserver uses as an input principally the elemental and organic composition and offers input files needed to run molecular dynamic (MD) simulations of solvated and neutralized SOM within the framework of the GROMOS 54A7 forcefield and the GROMOS and GROMACS simulation packages.

Orientational and Folding Thermodynamics via Electric Dipole Moment Restraining.

The projection of molecular processes onto a small set of relevant descriptors, the so-called reaction coordinates or collective variables (CVs), is a technique nowadays routinely employed by the biomolecular simulation community. In this work, we implemented two CVs to manipulate the orientation (i.e., angle) (µâƒ—a) and magnitude (|µâƒ—|) of the electric dipole moment. In doing so, we studied the thermodynamics of water orientation under the application of external voltages and the folding of two polypeptides at zero-field conditions. The projection of the free-energy [potential of mean force (PMF)] along water orientation defined an upper limit of around 0.3 V for irrelevant thermodynamic effects. On the other hand, sufficiently strong µâƒ—a restraints applied on 12-alanine (Ala12) triggered structural effects because of the alignment of local dipoles; for lower restraints, a full-body rotation is achieved. The manipulation of |µâƒ—| produced strong perturbations on the secondary structure of Ala12, promoting an enhanced sampling to its configurational space. Rigorous free-energy calculations in the form of 2-D PMFs for deca-alanine showed the utility of |µâƒ—| as a reaction coordinate to study folding in small α helices. As a whole, we propose that the manipulation of both components of the dipole moment, µâƒ—a and |µâƒ—|, provides thermodynamics insights into the structural conformation and stability of biomolecules. These new CVs are implemented in the Colvars module, available for NAMD and LAMMPS.

On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells.

Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Exploring the Membrane Potential of Simple Dual-Membrane Systems as Models for Gap-Junction Channels.

The conductance of ion channels can be modulated by a transmembrane potential difference, due to alterations on ion-mobility and also by changes in the pore structure. Despite the vast knowledge regarding the influence of voltage on transport properties of ion channels, little attention has been paid to describe, with atomic detail, the modulation of ionic transport in gap-junction channels (GJCs). Hence, molecular dynamics simulations were performed to explore the conductance of simple dual-membrane systems that account for the very basic features of GJCs. In doing so, we studied the influence of different charge distributions in the channel surface on these idealized systems under external electric fields, paying attention to the behavior of the electrostatic potential, ion density, ion currents, and equilibrium properties. Our results demonstrate that the incorporation of a charge distribution akin GJCs decreased anionic currents, favoring the transport of cationic species. Moreover, a thermodynamic characterization of ionic transport in these systems demonstrate the existence of a kinetic barrier that hinders anionic currents, reinforcing the role played by the internal arrangement of charges in GJCs. Overall, our results provide insights at the atomic scale on the effects of charge distributions over ionic transport, constituting a step forward into a better understanding of GJCs.

Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.

Characterization of a novel water pocket inside the human Cx26 hemichannel structure.

Connexins (Cxs) are a family of vertebrate proteins constituents of gap junction channels (GJCs) that connect the cytoplasm of adjacent cells by the end-to-end docking of two Cx hemichannels. The intercellular transfer through GJCs occurs by passive diffusion allowing the exchange of water, ions, and small molecules. Despite the broad interest to understand, at the molecular level, the functional state of Cx-based channels, there are still many unanswered questions regarding structure-function relationships, perm-selectivity, and gating mechanisms. In particular, the ordering, structure, and dynamics of water inside Cx GJCs and hemichannels remains largely unexplored. In this work, we describe the identification and characterization of a believed novel water pocket-termed the IC pocket-located in-between the four transmembrane helices of each human Cx26 (hCx26) monomer at the intracellular (IC) side. Using molecular dynamics (MD) simulations to characterize hCx26 internal water structure and dynamics, six IC pockets were identified per hemichannel. A detailed characterization of the dynamics and ordering of water including conformational variability of residues forming the IC pockets, together with multiple sequence alignments, allowed us to propose a functional role for this cavity. An in vitro assessment of tracer uptake suggests that the IC pocket residue Arg-143 plays an essential role on the modulation of the hCx26 hemichannel permeability.