Pornthep Sompornpisut

Open channel structure of MscL and the gating mechanism of mechanosensitive channels

Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin–spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 Å, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.

Structure of the KcsA channel intracellular gate in the open state.

Ion channels catalyze the selective transfer of ions across the membrane in response to a variety of stimuli. These channels gate by controlling the access of ions to a centrally located water-filled pore. The crystal structure of the Streptomyces lividans potassium channel (KcsA) has allowed a molecular exploration of this mechanism. Electron paramagnetic resonance (EPR) studies have uncovered significant conformational changes at the intracellular end of the second transmembrane helix (TM2) upon gating. We have used site-directed spin labeling (SDSL) and EPR spectroscopy in an attempt to quantify the structural rearrangements of the KcsA TM2 bundle underlying the transition from the closed to the open state. Under conditions favoring the closed and open conformations, 10 intersubunit distances were obtained across TM2 segments from tandem dimer constructs. Analysis of these data points to a mechanism in which each TM2 helix tilts away from the permeation pathway, towards the membrane plane, and rotates about its helical axis, supporting a scissoring-type motion with a pivot point near residues 107–108. These movements are accompanied by a large increase in the diameter of the vestibule below the central water-filled cavity.

Susceptibility of antiviral drugs against 2009 influenza A (H1N1) virus

The recent outbreak of the novel strain of influenza A (H1N1) virus has raised a global concern of the future risk of a pandemic. To understand at the molecular level how this new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely to already be resistant to, homology modeling and MD simulations have been applied on the H1N1 neuraminidase complexed with oseltamivir, and the M2-channel with adamantanes bound. The H1N1 virus was predicted to be susceptible to oseltamivir, with all important interactions with the binding residues being well conserved. In contrast, adamantanes are not predicted to be able to inhibit the M2 function and have completely lost their binding with the M2 residues. This is mainly due to the fact that the M2 transmembrane of the new H1N1 strain contains the S31N mutation which is known to confer resistance to adamantanes.

How amantadine and rimantadine inhibit proton transport in the M2 protein channel

To understand how antiviral drugs inhibit the replication of influenza A virus via the M2 ion channel, molecular dynamics simulations have been applied to the six possible protonation states of the M2 ion channel in free form and its complexes with two commercial drugs in a fully hydrated lipid bilayer. Among the six different states of free M2 tetramer, water density was present in the pore of the systems with mono-protonated, di-protonated at adjacent position, tri-protonated and tetra-protonated systems. In the presence of inhibitor, water density in the channel was considerably better reduced by rimantadine than amantadine, agreed well with the experimental IC(50) values. With the preferential position and orientation of the two drugs in all states, two mechanisms of action, where the drug binds to the opening pore and the histidine gate, were clearly explained, i.e., (i) inhibitor was detected to localize slightly closer to the histidine gate and can facilitate the orientation of His37 imidazole rings to lie in the close conformation and (ii) inhibitor acts as a blocker, binding at almost above the opening pore and interacts slightly with the three pore-lining residues, Leu26, Ala30 and Ser31. Here, the inhibitors were found to bind very weakly to the channel due to their allosteric hindrance while theirs side chains were strongly solvated.

The activated state of a sodium channel voltage sensor in a membrane environment

Direct structural insights on the fundamental mechanisms of permeation, selectivity, and gating remain unavailable for the Na+ and Ca2+ channel families. Here, we report the spectroscopic structural characterization of the isolated Voltage-Sensor Domain (VSD) of the prokaryotic Na+ channel NaChBac in a lipid bilayer. Site-directed spin-labeling and EPR spectroscopy were carried out for 118 mutants covering all of the VSD. EPR environmental data were used to unambiguously assign the secondary structure elements, define membrane insertion limits, and evaluate the activated conformation of the isolated-VSD in the membrane using restrain-driven molecular dynamics simulations. The overall three-dimensional fold of the NaChBac-VSD closely mirrors those seen in KvAP, Kv1.2, Kv1.2-2.1 chimera, and MlotiK1. However, in comparison to the membrane-embedded KvAP-VSD, the structural dynamics of the NaChBac-VSD reveals a much tighter helix packing, with subtle differences in the local environment of the gating charges and their interaction with the rest of the protein. Using cell complementation assays we show that the NaChBac-VSD can provide a conduit to the transport of ions in the resting or “down” conformation, a feature consistent with our EPR water accessibility measurements in the activated or “up” conformation. These results suggest that the overall architecture of VSD’s is remarkably conserved among K+ and Na+ channels and that pathways for gating-pore currents may be intrinsic to most voltage-sensors. Cell complementation assays also provide information about the putative location of the gating charges in the “down/resting” state and hence a glimpse of the extent of conformational changes during activation.

Long Time Scale GPU Dynamics Reveal the Mechanism of Drug Resistance of the Dual Mutant I223R/H275Y Neuraminidase from H1N1-2009 Influenza Virus

Multidrug resistance of the pandemic H1N1-2009 strain of influenza has been reported due to widespread treatment using the neuraminidase (NA) inhibitors, oseltamivir (Tamiflu), and zanamivir (Relenza). From clinical data, the single I223R (IR(1)) mutant of H1N1-2009 NA reduced efficacy of oseltamivir and zanamivir by 45 and 10 times, (1) respectively. More seriously, the efficacy of these two inhibitors against the double mutant I223R/H275Y (IRHY(2)) was significantly reduced by a factor of 12 374 and 21 times, respectively, compared to the wild-type.(2) This has led to the question of why the efficacy of the NA inhibitors is reduced by the occurrence of these mutations and, specifically, why the efficacy of oseltamivir against the double mutant IRHY was significantly reduced, to the point where oseltamivir has become an ineffective treatment. In this study, 1 μs of molecular dynamics (MD) simulations was performed to answer these questions. The simulations, run using graphical processors (GPUs), were used to investigate the effect of conformational change upon binding of the NA inhibitors oseltamivir and zanamivir in the wild-type and the IR and IRHY mutant strains. These long time scale dynamics simulations demonstrated that the mechanism of resistance of IRHY to oseltamivir was due to the loss of key hydrogen bonds between the inhibitor and residues in the 150-loop. This allowed NA to transition from a closed to an open conformation. Oseltamivir binds weakly with the open conformation of NA due to poor electrostatic interactions between the inhibitor and the active site. The results suggest that the efficacy of oseltamivir is reduced significantly because of conformational changes that lead to the open form of the 150-loop. This suggests that drug resistance could be overcome by increasing hydrogen bond interactions between NA inhibitors and residues in the 150-loop, with the aim of maintaining the closed conformation, or by designing inhibitors that can form a hydrogen bond to the mutant R223 residue, thereby preventing competition between R223 and R152.

Evolution of Human Receptor Binding Affinity of H1N1 Hemagglutinins from 1918 to 2009 Pandemic Influenza A Virus

The recent outbreak of the novel 2009 H1N1 influenza in humans has focused global attention on this virus, which could potentially have introduced a more dangerous pandemic of influenza flu. In the initial step of the viral attachment, hemagglutinin (HA), a viral glycoprotein surface, is responsible for the binding to the human SIA alpha2,6-linked sialopentasaccharide host cell receptor (hHAR). Dynamical and structural properties, based on molecular dynamics simulations of the four different HAs of Spanish 1918 (H1-1918), swine 1930 (H1-1930), seasonal 2005 (H1-2005), and a novel 2009 (H1-2009) H1N1 bound to the hHAR were compared. In all four HA-hHAR complexes, major interactions with the receptor binding were gained from HA residue Y95 and the conserved HA residues of the 130-loop, 190-helix, and 220-loop. However, introduction of the charged HA residues K145 and E227 in the 2009 HA binding pocket was found to increase the HA-hHAR binding efficiency in comparison to the three previously recognized H1N1 strains. Changing of the noncharged HA G225 residue to a negatively charged D225 provides a larger number of hydrogen-bonding interactions. The increase in hydrophilicity of the receptor binding region is apparently an evolution of the current pandemic flu from the 1918 Spanish, 1930 swine, and 2005 seasonal strains. Detailed analysis could help the understanding of how different HAs effectively attach and bind with the hHAR.

Molecular Mechanism of Mg2+-dependent gating in CorA

CorA is the major transport system responsible for Mg2+ uptake in bacteria and can functionally substitute for its homologue Mrs2p in the yeast inner mitochondrial membrane. Although several CorA crystal structures are available, the molecular mechanism of Mg2+ uptake remains to be established. Here we use EPR spectroscopy, electrophysiology and molecular dynamic simulations to show that CorA is regulated by cytoplasmic Mg2+ acting as a ligand and elucidate the basic conformational rearrangements responsible for Mg2+-dependent gating. Mg2+ unbinding at the divalent cation sensor triggers a conformational change that leads to the inward motion of the stalk helix, which propagates to the pore forming transmembrane helix TM1. Helical tilting and rotation in TM1 generates an iris-like motion that increases the diameter of the permeation pathway, triggering ion conduction. This work establishes the molecular basis of a Mg2+-driven negative feedback loop in CorA as the key physiological event controlling Mg2+ uptake and homeostasis in prokaryotes.

Why amantadine loses its function in influenza m2 mutants: MD simulations.

Molecular dynamics simulations of the drug-resistant M2 mutants, A30T, S31N, and L26I, were carried out to investigate the inhibition of M2 activity using amantadine (AMT). The closed and open channel conformations were examined via non- and triply protonated H37. For the nonprotonated state, these mutants exhibited zero water density in the conducting region, and AMT was still bound to the channel pore. Thus, water transport is totally suppressed, similar to the wild-type channel. In contrast, the triply protonated states of the mutants exhibited a different water density and AMT position. A30T and L26I both have a greater water density compared to the wild-type M2, while for the A30T system, AMT is no longer inside the pore. Hydrogen bonding between AMT and H37 crucial for the bioactivity is entirely lost in the open conformation. The elimination of this important interaction of these mutations is responsible for the lost of AMTs function in influenza A M2. This is different for the S31N mutant in which AMT was observed to locate at the pore opening region and bond with V27 instead of S31.

Source of High Pathogenicity of an Avian Influenza Virus H5N1: Why H5 Is Better Cleaved by Furin

The origin of the high pathogenicity of an emerging avian influenza H5N1 due to the -RRRKK- insertion at the cleavage loop of the hemagglutinin H5, was studied using the molecular dynamics technique, in comparison with those of the noninserted H5 and H3 bound to the furin (FR) active site. The cleavage loop of the highly pathogenic H5 was found to bind strongly to the FR cavity, serving as a conformation suitable for the proteolytic reaction. With this configuration, the appropriate interatomic distances were found for all three reaction centers of the enzyme-substrate complex: the arrangement of the catalytic triad, attachment of the catalytic Ser(368) to the reactive S1-Arg, and formation of the oxyanion hole. Experimentally, the--RRRKK--insertion was also found to increase in cleavage of hemagglutinin by FR. The simulated data provide a clear answer to the question of why inserted H5 is better cleaved by FR than the other subtypes, explaining the high pathogenicity of avian influenza H5N1.