Victoria Oakes

Lateral Fenestrations in K+-Channels Explored Using Molecular Dynamics Simulations

Potassium channels are of paramount physiological and pathological importance and therefore constitute significant drug targets. One of the keys to rationalize the way drugs modulate ion channels is to understand the ability of such small molecules to access their respective binding sites, from which they can exert an activating or inhibitory effect. Many computational studies have probed the energetics of ion permeation, and the mechanisms of voltage gating, but little is known about the role of fenestrations as possible mediators of drug entry in potassium channels. To explore the existence, structure, and conformational dynamics of transmembrane fenestrations accessible by drugs in potassium channels, molecular dynamics simulation trajectories were analyzed from three potassium channels: the open state voltage-gated channel Kv1.2, the G protein-gated inward rectifying channel GIRK2 (Kir3.2), and the human two-pore domain TWIK-1 (K2P1.1). The main results of this work were the identification of the sequence identity of four main lateral fenestrations of similar length and with bottleneck radius in the range of 0.9-2.4 Å for this set of potassium channels. It was found that the fenestrations in Kv1.2 and Kir3.2 remain closed to the passage of molecules larger than water. In contrast, in the TWIK-1 channel, both open and closed fenestrations are sampled throughout the simulation, with bottleneck radius shown to correlate with the random entry of lipid membrane molecules into the aperture of the fenestrations. Druggability scoring function analysis of the fenestration regions suggests that Kv and Kir channels studied are not druggable in practice due to steric constraining of the fenestration bottleneck. A high (>50%) fenestration sequence identity was found in each potassium channel subfamily studied, Kv1, Kir3, and K2P1. Finally, the reported fenestration sequence of TWIK-1 compared favorably with another channel, K2P channel TREK-2, reported to possess open fenestrations, suggesting that K2P channels could be druggable via fenestrations, for which we reported atomistic detail of the fenestration region, including the flexible residues M260 and L264 that interact with POPC membrane in a concerted fashion with the aperture and closure of the fenestrations.

Voltage-Gated Sodium Channels: Mechanistic Insights From Atomistic Molecular Dynamics Simulations

The permeation of ions and other molecules across biological membranes is an inherent requirement of all cellular organisms. Ion channels, in particular, are responsible for the conduction of charged species, hence modulating the propagation of electrical signals. Despite the universal physiological implications of this property, the molecular functioning of ion channels remains ambiguous. The combination of atomistic structural data with computational methodologies, such as molecular dynamics (MD) simulations, is now considered routine to investigate structure-function relationships in biological systems. A fuller understanding of conduction, selectivity, and gating, therefore, is steadily emerging due to the applicability of these techniques to ion channels. However, because their structure is known at atomic resolution, studies have consistently been biased toward K(+) channels, thus the molecular determinants of ionic selectivity, activation, and drug blockage in Na(+) channels are often overlooked. The recent increase of available crystallographic data has eminently encouraged the investigation of voltage-gated sodium (NaV) channels via computational methods. Here, we present an overview of simulation studies that have contributed to our understanding of key principles that underlie ionic conduction and selectivity in Na(+) channels, in comparison to the K(+) channel analogs.

Exploring the Dynamics of the TWIK-1 Channel

Potassium channels in the two-pore domain family (K2P) have various structural attributes that differ from those of other K+ channels, including a dimeric assembly constituted of nonidentical domains and an expansive extracellular cap. Crystallization of the prototypical K2P channel, TWIK-1, finally revealed the structure of these characteristics in atomic detail, allowing computational studies to be undertaken. In this study, we performed molecular-dynamics simulations for a cumulative time of ∼1 μs to discern the mechanism of ion transport throughout TWIK-1. We observed the free passage of ions beneath the extracellular cap and identified multiple high-occupancy sites in close proximity to charged residues on the protein surface. Despite the overall topological similarity of the x-ray structure of the selectivity filter to other K+ channels, the structure diverges significantly in molecular-dynamics simulations as a consequence of nonconserved residues in both pore domains contributing to the selectivity filter (T118 and L228). The behavior of such residues has been linked to channel inactivation and the phenomenon of dynamic selectivity, where TWIK-1 displays robust Na+ inward flux in response to subphysiological K+ concentrations.

Voltage-Gated Sodium Channels

The permeation of ions and other molecules across biological membranes is an inherent requirement of all cellular organisms. Ion channels, in particular, are responsible for the conduction of charged species, hence modulating the propagation of electrical signals. Despite the universal physiological implications of this property, the molecular functioning of ion channels remains ambiguous. The combination of atomistic structural data with computational methodologies, such as molecular dynamics (MD) simulations, is now considered routine to investigate structure-function relationships in biological systems. A fuller understanding of conduction, selectivity, and gating, therefore, is steadily emerging due to the applicability of these techniques to ion channels. However, because their structure is known at atomic resolution, studies have consistently been biased toward K(+) channels, thus the molecular determinants of ionic selectivity, activation, and drug blockage in Na(+) channels are often overlooked. The recent increase of available crystallographic data has eminently encouraged the investigation of voltage-gated sodium (NaV) channels via computational methods. Here, we present an overview of simulation studies that have contributed to our understanding of key principles that underlie ionic conduction and selectivity in Na(+) channels, in comparison to the K(+) channel analogs.

Stereospecific Interactions of Cholesterol in a Model Cell Membrane: Implications for the Membrane Dipole Potential

Cholesterol is a major constituent of the plasma membrane in higher order eukaryotes. The effect of cholesterol on the structure and organisation of cell membranes has been studied extensively by both experimental and computational means. In recent years, a wealth of data has been accumulated illustrating how subtle differences in the structure of cholesterol equate to considerable changes in the physical properties of the membrane. The effect of cholesterol stereoisomers, in particular, has been established, identifying a direct link with the activity of specific membrane proteins. In this study, we perform extensive molecular dynamics simulations of phospholipid bilayers containing three isomers of cholesterol, the native form (nat-cholesterol), the enantiomer of the native form (ent-cholesterol), and an epimer of cholesterol that differs by the orientation of the polar hydroxyl group (epi-cholesterol). Based on these simulations, an atomic-level description of the stereospecific cholesterol–phospholipid interactions is provided, establishing a potential mechanism for the perturbation of membrane properties, specifically the membrane dipole potential.

Capturing the Molecular Mechanism of Anesthetic Action by Simulation Methods

Significant computational efforts have been focused toward exposing the molecular mechanisms of anesthesia in recent years. In the past decade, this has been aided considerably by a momentous increase in the number of high-resolution structures of ion channels, which are putative targets for the anesthetic agents, as well as advancements in high-performance computing technologies. In this review, typical simulation methods to investigate the behavior of model membranes and membrane-protein systems are briefly reviewed, and related computational studies are surveyed. Both lipid- and protein-mediated mechanisms of anesthetic action are scrutinized, focusing on the behavior of ion channels in the latter case.

Exposure of the HIV-1 broadly neutralizing antibody 10E8 MPER epitope on the membrane surface by gp41 transmembrane domain scaffolds

The 10E8 antibody achieves near-pan neutralization of HIV-1 by targeting the remarkably conserved gp41 membrane-proximal external region (MPER) and the connected transmembrane domain (TMD) of the HIV-1 envelope glycoprotein (Env). Thus, recreating the structure that generates 10E8-like antibodies is a major goal of the rational design of anti-HIV vaccines. Unfortunately, high-resolution information of this segment in the native Env is lacking, limiting our understanding of the behavior of the crucial 10E8 epitope residues. In this report, two sequences, namely, MPER-TMD1 (gp41 residues 671-700) and MPER-TMD2 (gp41 residues 671-709) were compared both experimentally and computationally, to assess the TMD as a potential membrane integral scaffold for the 10E8 epitope. These sequences were selected to represent a minimal (MPER-TMD1) or full-length (MPER-TMD2) TMD membrane anchor according to mutagenesis results reported by Yue et al. (2009) J. Virol. 83, 11,588. Immunochemical assays revealed that MPER-TMD1, but not MPER-TMD2, effectively exposed the MPER C-terminal stretch, harboring the 10E8 epitope on the surface of phospholipid bilayers containing a cholesterol concentration equivalent to that of the viral envelope. Molecular dynamics simulations, using the recently resolved TMD trimer structure combined with the MPER in a cholesterol-enriched model membrane confirmed these results and provided an atomistic mechanism of epitope exposure which revealed that TMD truncation at position A700 combined with N-terminal addition of lysine residues positively impacts epitope exposure. Overall, these results provide crucial insights into the design of effective MPER-TMD derived immunogens.

Computer Simulations of Membrane Proteins

Developments in computational algorithms and structure-determination technologies have enabled molecular dynamics simulations to become a routine tool to investigate the structure and dynamics of biological membranes and membrane proteins in great detail. In this chapter, we provide an overview of atomistic molecular dynamics simulations and related methods, such as coarse-grain simulations and biased sampling methods, and illustrate using key examples how such methods have advanced our understanding in the field of membrane protein biophysics. We exemplify how MD simulations have provided insights into selective permeation mechanisms through lipid bilayers and ion channels, as well as conformational changes associated with transport in both G-protein coupled receptors and membrane transporters.

Chapter 10:Novel Insights into Membrane Transport from Computational Methodologies

Atomic-resolution imaging of the plasma membrane and its constituents has advanced significantly in recent years. However, membrane transport is profoundly reliant on dynamic processes ranging from highly concerted atomic fluctuations to large-scale conformational changes, which cannot be sufficiently described by static structural information. As a consequence, computational methodologies have become a prominent tool for investigating membrane organisation and dynamics. In particular, molecular dynamics simulation has proven to be a pertinent method for investigating how matter is transported through membranes, either directly through the membrane or via integral membrane proteins, in an appropriate level of detail. In this chapter, a brief overview of molecular dynamics simulations and related methodologies will be provided, and use prototypical biological systems to illustrate how these methods have contributed to our understanding of unassisted diffusion through membranes, passive diffusion through ion channels, signalling through receptors and active transport through transporters.

Chapter 2: Molecular Dynamics Simulations: Principles and Applications for the Study of Membrane Proteins

The plasma membrane is responsible for the maintenance of the correct chemical composition in cells, separating harmful substances from key biochemical processes required for basic human function. Membrane proteins are responsible for communication and transport phenomena across the membrane, facilitating a dynamic relationship between the cell interior and exterior despite the physical blockade. How these proteins function on a molecular level, however, remains largely unresolved. A fuller understanding is steadily emerging due to the increasing availability of three-dimensional structures of membrane proteins, in combination with computational methodologies such as molecular dynamics simulations. In this chapter, we present the key principles and considerations of performing molecular dynamics simulations in the context of membrane proteins, highlighting the leading applications in this field.