Conformational Selection of a Tryptophan Side Chain Drives the Generalized Increase in Activity of PET Hydrolases through a Ser/Ile Double Mutation.

Poly(ethylene terephthalate) (PET) is the most common polyester plastic in the packaging industry and a major source of environmental pollution due to its single use. Several enzymes, termed PET hydrolases, have been found to hydrolyze this polymer at different temperatures, with the enzyme from Ideonella sakaiensis (IsPETase) having optimal catalytic activity at 30-35 °C. Crystal structures of IsPETase have revealed that the side chain of a conserved tryptophan residue within an active site loop (W185) shifts between three conformations to enable substrate binding and product release. This is facilitated by two residues unique to IsPETase, S214 and I218. When these residues are inserted into other PET hydrolases in place of the otherwise strictly conserved histidine and phenylalanine residues found at their respective positions, they enhance activity and decrease T opt. Herein, we combine molecular dynamics and well-tempered metadynamics simulations to investigate dynamic changes of the S214/I218 and H214/F218 variants of IsPETase, as well as three other mesophilic and thermophilic PET hydrolases, at their respective temperature and pH optima. Our simulations show that the S214/I218 insertion both increases the flexibility of active site loop regions harboring key catalytic residues and the conserved tryptophan and expands the conformational plasticity of this tryptophan side chain, enabling the conformational transitions that allow for substrate binding and product release in IsPETase. The observed catalytic enhancement caused by this substitution in other PET hydrolases appears to be due to conformational selection, by capturing the conformational ensemble observed in IsPETase.

Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling.

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism.

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC-the 5-HT3A receptor-unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.

Cholesterol content in the membrane promotes key lipid-protein interactions in a pentameric serotonin-gated ion channel.

Pentameric ligand-gated ion channels (pLGICs), embedded in the lipid membranes of nerve cells, mediate fast synaptic transmission and are major pharmaceutical targets. Because of their complexity and the limited knowledge of their structure, their working mechanisms have still to be fully unraveled at the molecular level. Over the past few years, evidence that the lipid membrane may modulate the function of membrane proteins, including pLGICs, has emerged. Here, we investigate, by means of molecular dynamics simulations, the behavior of the lipid membrane at the interface with the 5-HT3A receptor (5-HT3AR), a representative pLGIC which is the target of nausea-suppressant drugs, in a nonconductive state. Three lipid compositions are studied, spanning different concentrations of the phospholipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and of cholesterol, hence a range of viscosities. A variety of lipid interactions and persistent binding events to different parts of the receptor are revealed in the investigated models, providing snapshots of the dynamical environment at the membrane-receptor interface. Some of these events result in lipid intercalation within the transmembrane domain, and others reach out to protein key sections for signal transmission and receptor activation, such as the Cys-loop and the M2-M3 loop. In particular, phospholipids, with their long hydrophobic tails, play an important role in these interactions, potentially providing a bridge between these two structures. A higher cholesterol content appears to promote lipid persistent binding to the receptor.

Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer.

Förster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (〈EFRETexp〉 = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (〈EFRETMD〉 = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors 〈κ2〉 = 0.17 ± 0.16 and 〈|κ|〉 = 0.35 ± 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time- and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET.

Mutagenesis computer experiments in pentameric ligand-gated ion channels: the role of simulation tools with different resolution.

Pentameric ligand-gated ion channels (pLGICs) are an important class of widely expressed membrane neuroreceptors, which play a crucial role in fast synaptic communications and are involved in several neurological conditions. They are activated by the binding of neurotransmitters, which trigger the transmission of an electrical signal via facilitated ion flux. They can also be activated, inhibited or modulated by a number of drugs. Mutagenesis electrophysiology experiments, with natural or unnatural amino acids, have provided a large body of functional data that, together with emerging structural information from X-ray spectroscopy and cryo-electron microscopy, are helping unravel the complex working mechanisms of these neuroreceptors. Computer simulations are complementing these mutagenesis experiments, with insights at various levels of accuracy and resolution. Here, we review how a selection of computational tools, including first principles methods, classical molecular dynamics and enhanced sampling techniques, are contributing to construct a picture of how pLGICs function and can be pharmacologically targeted to treat the disorders they are responsible for.

Trans- Cis Proline Switches in a Pentameric Ligand-Gated Ion Channel: How They Are Affected by and How They Affect the Biomolecular Environment.

Pentameric ligand-gated ion channels (pLGICs) are important neuroreceptors, embedded in neuronal membranes, that mediate fast synaptic transmission. The molecular details of their working mechanisms have still to be fully unravelled due to their complexity and limited structural information available. Here we focus on a potential molecular switch in a prototypical pLGIC, the serotonin-activated 5-HT3 receptor, consisting of the trans- cis isomerization of a proline at the interface between the extracellular and transmembrane domain. Mutagenesis electrophysiology experiments previously showed that if such isomerization could not take place, the channel would not open, but the hypothetical role of this mechanism as key to channel gating is still debated. We investigate this switch within the receptor with molecular dynamics and enhanced sampling simulations. We analyze how the isomerization free energy landscape is affected by the receptor environment in comparison to simplified models. Moreover, we reveal how the isomerization, in turn, affects the structural and electrostatic properties of the receptor at the extracellular-transmembrane domain interface, e.g., by tuning the ion selectivity filter.