Interactions of Na(+), K(+), Rb(+), and Cs(+) ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K(+) and Rb(+), are similar in their interactions with the selectivity filter. In contrast, Na(+) ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs(+) ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs(+) ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.

Molecular dynamics (MD) simulations have been used to unmask details of specific interactions of anionic phospholipids with intersubunit binding sites on the surface of the bacterial potassium channel KcsA. Crystallographic data on a diacyl glycerol fragment at this site were used to model phosphatidylethanolamine (PE), or phosphatidylglycerol (PG), or phosphatidic acid (PA) at the intersubunit binding sites. Each of these models of a KcsA-lipid complex was embedded in phosphatidyl choline bilayer and explored in a 20 ns MD simulation. H-bond analysis revealed that in terms of lipid-protein interactions PA > PG >> PE and revealed how anionic lipids (PG and PA) bind to a site provided by two key arginine residues (R(64) and R(89)) at the interface between adjacent subunits. A 27 ns simulation was performed in which KcsA (without any lipids initially modeled at the R(64)/R(89) sites) was embedded in a PE/PG bilayer. There was a progressive specific increase over the course of the simulation in the number of H-bonds of PG with KcsA. Furthermore, two specific PG binding events at R(64)/R(89) sites were observed. The phosphate oxygen atoms of bound PG formed H-bonds to the guanidinium group of R(89), whereas the terminal glycerol H-bonded to R(64). Overall, this study suggests that simulations can help identify and characterize sites for specific lipid interactions on a membrane protein surface.

Experimental evidence has shown a close correlation between the composition and physical state of the membrane bilayer and glucose transport activity via the glucose transporter GLUT1. Cooling alters the membrane lipids from the fluid to gel phase, and also causes a large decrease in the net glucose transport rate. The goal of this study is to investigate how the physical phase of the membrane alters glucose transporter structural dynamics using molecular-dynamics simulations. Simulations from an initial fluid to gel phase reduce the size of the cavities and tunnels traversing the protein and connecting the external regions of the transporter and the central binding site. These effects can be ascribed solely to membrane structural changes since in silico cooling of the membrane alone, while maintaining the higher protein temperature, shows protein structural and dynamic changes very similar to those observed with uniform cooling. These results demonstrate that the protein structure is sensitive to the membrane phase, and have implications for how transmembrane protein structures respond to their physical environment.

Ion channels enable diffusion of ions down physiological electrochemical gradients. Modulation of ion permeation is crucial for the physiological functioning of cells, and misregulation of ion channels is linked to a myriad of channelopathies. The ion permeation mechanism in the transient receptor potential (TRP) ion channel family is currently not understood at an atomistic level. In this work, we employed a simulation strategy for ion permeation (molecular-dynamics simulations with bias-exchange metadynamics) to study and compare monovalent (Na(+), K(+)) ion permeation in the open-activated TRP vanniloid-1 (TRPV1) ion channel. Using ∼3.6 μs of simulation trajectories, we obtained atomistic evidence for the nonselective nature of TRPV1. Our analysis shows that solvated monovalent ions permeate through the selectivity filter with comparable energetic barriers via a two-site mechanism. Finally, we confirmed that an intracellular binding site is located between the intracellular gate residues I679 and E684.

The interactions between membrane proteins and their lipid bilayer environment play important roles in the stability and function of such proteins. Extended (15-20 ns) molecular dynamics simulations have been used to explore the interactions of two membrane proteins with phosphatidylcholine bilayers. One protein (KcsA) is an alpha-helix bundle and embedded in a palmitoyl oleoyl phosphatidylcholine bilayer; the other (OmpA) is a beta-barrel outer-membrane protein and is in a dimyristoyl phosphatidylcholine bilayer. The simulations enable analysis in detail of a number of aspects of lipid-protein interactions. In particular, the interactions of aromatic amphipathic side chains (i.e., Trp, Tyr) with lipid headgroups, and "snorkeling" interactions of basic side chains (i.e., Lys, Arg) with phosphate groups are explored. Analysis of the number of contacts and of H-bonds reveal fluctuations on an approximately 1- to 5-ns timescale. There are two clear bands of interacting residues on the surface of KcsA, whereas there are three such bands on OmpA. A large number of Arg-phosphate interactions are seen for KcsA; for OmpA, the number of basic-phosphate interactions is smaller and shows more marked fluctuations with respect to time. Both classes of interaction occur in clearly defined interfacial regions of width approximately 1 nm. Analysis of lateral diffusion of lipid molecules reveals that "boundary" lipid molecules diffuse at about half the rate of bulk lipid. Overall, these simulations present a dynamic picture of lipid-protein interactions: there are a number of more specific interactions but even these fluctuate on an approximately 1- to 5-ns timescale.

The bacterial channel KirBac1.1 provides a structural homolog of mammalian inward rectifier potassium (Kir) channels. The conformational dynamics of the selectivity filter of Kir channels are of some interest in the context of possible permeation and gating mechanisms for this channel. Molecular dynamics simulations of KirBac have been performed on a 10-ns timescale, i.e., comparable to that of ion permeation. The results of five simulations (total simulation time 50 ns) based on three different initial ion configurations and two different model membranes are reported. These simulation data provide evidence for limited (<0.1 nm) filter flexibility during the concerted motion of ions and water molecules within the filter, such local changes in conformation occurring on an approximately 1-ns timescale. In the absence of K(+) ions, the KirBac selectivity filter undergoes more substantial distortions. These resemble those seen in comparable simulations of other channels (e.g., KcsA and KcsA-based homology models) and are likely to lead to functional closure of the channel. This suggests filter distortions may provide a mechanism of K-channel gating in addition to changes in the hydrophobic gate formed at the intracellular crossing point of the M2 helices. The simulation data also provide evidence for interactions of the "slide" (pre-M1) helix of KirBac with phospholipid headgroups.

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.