Myunggi Yi

Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer.

M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric proton channel in the viral envelope and is essential for viral replication. Two manuscripts shed light on the functional mechanism of this channel. Sharma et al. (p. 509; see the Perspective by Fiorin et al.) determined the structure of the conductance domain in a lipid bilayer and propose that a histidine and tryptophan from each monomer form a cluster that guides protons through the channel in a mechanism that involves forming and breaking hydrogen bonds between adjacent pairs of histidines. Hu et al. (p. 505; see the Perspective by Fiorin et al.) focused on the structure and dynamics of the proton-selective histidine at high and low pH, proposing that proton conduction involves histidine deprotonation and reprotonation. A tetrameric cluster of histidine and tryptophan residues, through its unique chemistry, shepherds protons through the M2 channel. The M2 protein from the influenza A virus, an acid-activated proton-selective channel, has been the subject of numerous conductance, structural, and computational studies. However, little is known at the atomic level about the heart of the functional mechanism for this tetrameric protein, a His37-Trp41 cluster. We report the structure of the M2 conductance domain (residues 22 to 62) in a lipid bilayer, which displays the defining features of the native protein that have not been attainable from structures solubilized by detergents. We propose that the tetrameric His37-Trp41 cluster guides protons through the channel by forming and breaking hydrogen bonds between adjacent pairs of histidines and through specific interactions of the histidines with the tryptophan gate. This mechanism explains the main observations on M2 proton conductance.

Influence of Solubilizing Environments on Membrane Protein Structures

Membrane protein structures are stabilized by weak interactions and are influenced by additional interactions with the solubilizing environment. Structures of influenza virus A M2 protein, a proven drug target, have been determined in three different environments, thus providing a unique opportunity to assess environmental influences. Structures determined in detergents and detergent micelles can have notable differences from those determined in lipid bilayers. These differences make it imperative to validate membrane protein structures.

A secondary gate as a mechanism for inhibition of the M2 proton channel by amantadine.

The mechanism of inhibition of the influenza A virus M2 proton channel by the antiviral drug amantadine has been under intense investigation. The importance of a mechanistic understanding is heightened by the prevalence of amantadine-resistant mutations. To gain mechanistic insight at the molecular level, we carried out extensive molecular dynamics simulations of the tetrameric M2 proton channel in both apo and amantadine-bound forms in a lipid bilayer. The simulation of the apo form revealed that Val27 from the four M2 subunits can form a secondary gate near the channel entrance and break the water wire in the channel pore. This gate arises from physical occlusion and the elimination of hydrogen-bonding partners for water molecules. In the presence of amantadine, the secondary gate formed by Val27 and the drug molecule lying just below form an extended blockage, which breaks the water wire throughout the simulation. The location and orientation of amantadine inside of the channel pore as found in our simulation are supported by a host of experimental observations. Our study suggests a novel role for Val27 in the inhibition of the M2 proton channel by amantadine.

Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

The M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Structure determination by solid-state and solution NMR and X-ray crystallography has contributed significantly to our understanding, but channel activation may involve conformations not captured by these studies. Indeed, solid-state NMR data demonstrate that the M2 protein possesses significant conformational heterogeneity. Here, we report molecular dynamics (MD) simulations of the M2 transmembrane domain (TMD) in the absence and presence of the antiviral drug amantadine. The ensembles of MD conformations for both apo and bound forms reproduced the NMR data well. The TMD helix was found to kink around Gly-34, where water molecules penetrated deeply into the backbone. The amantadine-bound form exhibited a single peak ≈10° in the distribution of helix-kink angle, but the apo form exhibited 2 peaks, ≈0° and 40°. Conformations of the apo form with small and large kink angles had narrow and wide pores, respectively, around the primary gate formed by His-37 and Trp-41. We propose a structural model for channel activation, in which the small-kink conformations dominate before proton uptake by His-37 from the exterior, and proton uptake makes the large-kink conformations more favorable, thereby priming His-37 for proton release to the interior.

Spontaneous conformational change and toxin binding in α7 acetylcholine receptor: Insight into channel activation and inhibition

Nicotinic AChRs (nAChRs) represent a paradigm for ligand-gated ion channels. Despite intensive studies over many years, our understanding of the mechanisms of activation and inhibition for nAChRs is still incomplete. Here, we present molecular dynamics (MD) simulations of the α7 nAChR ligand-binding domain, both in apo form and in α-Cobratoxin-bound form, starting from the respective homology models built on crystal structures of the acetylcholine-binding protein. The toxin-bound form was relatively stable, and its structure was validated by calculating mutational effects on the toxin-binding affinity. However, in the apo form, one subunit spontaneously moved away from the conformation of the other four subunits. This motion resembles what has been proposed for leading to channel opening. At the top, the C loop and the adjacent β7-β8 loop swing downward and inward, whereas at the bottom, the F loop and the C terminus of β10 swing in the opposite direction. These swings appear to tilt the whole subunit clockwise. The resulting changes in solvent accessibility show strong correlation with experimental results by the substituted cysteine accessibility method upon addition of acetylcholine. Our MD simulation results suggest a mechanistic model in which the apo form, although predominantly sampling the “closed” state, can make excursions into the “open” state. The open state has high affinity for agonists, leading to channel activation, whereas the closed state upon distortion has high affinity for antagonists, leading to inhibition.

Blocking Effect of an Immuno-Suppressive Agent, Cynarin, on CD28 of T-Cell Receptor

PurposeCynarin, a potential immunosuppressant that blocks the interaction between the CD28 of T-cell receptor and CD80 of antigen presenting cells, was found in Echinacea purpurea by a new pharmaceutical screening method: After Flowing Through Immobilized Receptor (AFTIR; Dong et al., J Med Chem, 49: 1845-1854, 2006). This Echinacea component is the first small molecule that is able to specifically block “signal 2” of T-cell activation.MethodsIn this study, we used the AFTIR method to further confirm that cynarin effectively blocked the binding between CD80 of B-cells and CD28 of T-cells, and provide details of its mechanism of action.ResultsThe experimental results showed that cynarin blocked about 87% of the CD28-dependent “signal 2” pathway of T-cell activation under the condition of one to one ratio of T-cell and B-cell in vitro. Theoretical structure modeling showed that cynarin binds to the “G-pocket” of CD28 (Evans et al., Nat Immunol, 6:271-279, 2005), and thus interrupts the site of interaction between CD28 and CD80.ConclusionsThese results confirm both that AFTIR is a promising method for screening selective active compounds from herbal medicine and that cynarin has great potential as an immuno-suppressive agent.

Silver-Lactoferrin Nanocomplexes as a Potent Antimicrobial Agent

The process of silver immobilization onto and/or into bovine lactoferrin (LTF), the physicochemical properties of bovine lactoferrin and obtained silver-lactoferrin complexes, as well as antibacterial activity of silver-lactoferrin complexes were investigated in this work. Kinetic study of the silver immobilization into lactoferrin was carried out using batch sorption techniques. Spectrometric (MALDI-TOF/TOF-MS, ICP-MS), spectroscopic (FTIR, SERS), electron microscopic (TEM) and electrophoretic (I-DE) techniques, as well as zeta potential measurements, were applied for characterization of LTF and binding nature of silver in Ag-LTF complexes. On the basis of the results of the kinetics study, it was established that the silver binding to LTF is a heterogeneous process involving two main stages: (i) internal diffusion and sorption onto external surface of lactoferrin globules; and (ii) internal diffusion and binding into lactoferrin globule structure. Spectroscopic techniques combined with TEM analysis confirmed the binding process. Molecular dynamics (MD) analysis was carried out in order to simulate the mechanism of the binding process, and locate potential binding sites, as well as complement the experimental findings. Quantum mechanics (QM) simulations were performed utilizing density functional theory (DFT) in order to support the reduction mechanism of silver ions to elemental silver. Antimicrobial activity of synthesized lactoferrin complexes against selected clinical bacteria was confirmed using flow cytometry and antibiograms.

Ab initio calculations and validation of the pH-dependent structures of the His37-Trp41 quartet, the heart of acid activation and proton conductance in the M2 protein of Influenza A virus.

The M2 protein of Influenza A virus forms a homotetrameric proton channel activated by low pH. The His37-Trp41 quartet is the heart of acid activation and proton conductance, but the functional mechanism is still controversial. We carried out ab initio calculations to model the pH-dependent structures of the His37-Trp41 quartet. In our model at neutral pH, the four His37 residues are configured into a pair of dimers; in each dimer, a proton is shared between Nδ1 on one residue and Nε2 on the other, and, under the restraint of the backbone, the two imidazole rings are nearly parallel, in contrast to a perpendicular arrangement for a free imidazole-imidazolium dimer. Within each dimer the +1 charge is highly delocalized, contributing to its stabilization in a low dielectric environment. The Nδ1-H-Nε2 strong hydrogen bonds result in significantly downfield shifted Nδ1 and Nε2 chemical shifts (at 169.7 and 167.6 ppm, respectively), in good agreement with experiments. In our model at acidic pH (where the channel becomes activated), a third proton binds to an imidazole-imidazolium dimer; the imidazole rings rotate away (each by ~55°) from each other, destroying the dimer structure. The two imidazoliums are stabilized by hydrogen bonds with water molecules and a cation-π interaction with Trp41. The Raman spectra calculated for the His37-Trp41 quartet at neutral and acidic pH are in agreement with experiments. Our calculations support an activation and conductance mechanism in which a hydronium ion from the N-terminal side passes a proton to an imidazole-imidazolium dimer; when the Trp41 gate is open, relaying of a proton onto a water molecule from the C-terminal side then allows the imidazole-imidazolium dimer to reform and be ready for the next round of proton conductance.

Molecular cloning and characterization of three cDNAs encoding allatostatin-like neurosecretory peptides from Pandalopsis japonica.

Three cDNAs encoding allatostatin-like peptides (two myoinhibitory peptides; Paj-MIPI and Paj-MIPII, and one C-type AST; Paj-ASTC) were identified from Pandalopsis japonica through a combination of bioinformatic analysis and PCR-based gene cloning strategy. Paj-MIPI and Paj-MIPII encoded proteins with 189 and 117 amino acid residues, respectively, and a total of 10 mature peptides are putatively produced from the two MIP cDNAs (seven from Paj-MIPI and three from Paj-MIPII). Among the MIPs from various arthropods, their size and organization varied and it was unable to establish the monophyletic evolutionary relationship, which is mainly due to difference in the number and location of the mature peptide W(X(6))W motif of each MIP gene. Based on the sequence similarity of six residues flanked by two conserved tryptophan (W) residues, crustacean MIPs could be further classified into at least four groups. Paj-ASTC cDNA (648bp) encoded a protein with 143 amino acid residues. The prepropeptide of Paj-ASTC showed conserved C-type AST characteristics including a signal sequence, two dibasic cleavage sites, and a mature peptide sequence with two cysteine residues at the 7th and 14th positions, creating a disulfide bridge. Based on the sequence similarity in the mature peptides, the ASTCs in arthropods could be further classified into two subgroups, AVSCF-ASTC and PISCF-ASTC. Phylogenetic and sequence similarity analysis showed that Paj-ASTC belonged to the PISCF-ASTC subgroup. Expression studies revealed that AST-like peptides from P. japonica were mainly expressed in neuronal tissues, and the expression of Paj-ASTC was also detected in the intestine. Eyestalk ablation (ESA) altered the mRNA expression levels of both Paj-MIPs and Paj-ASTC, suggesting that factors from the sinus gland/X organ complex had a transient effect on AST-like peptide transcription. Correlation analysis of three allatostatin-like peptides revealed a strong positive correlation in brain tissues, suggesting that transcriptional regulation of three allatostatin-like peptides from P. japonica is influenced by the similar physiological condition.

Fish bone peptide promotes osteogenic differentiation of MC3T3-E1 pre-osteoblasts through upregulation of MAPKs and Smad pathways activated BMP-2 receptor

Fish bone, a by‐product of fishery processing, is composed of protein, calcium, and other minerals. The objective of this study was to investigate the effects of a bioactive peptide isolated from the bone of the marine fish, Johnius belengerii, on the osteoblastic differentiation of MC3T3‐E1 pre‐osteoblasts. Post consecutive purification by liquid chromatography, a potent osteogenic peptide, composed of 3 amino acids, Lys‐Ser‐Ala (KSA, MW: 304.17 Da), was identified. The purified peptide promoted cell proliferation, alkaline phosphatase activity, mineral deposition, and expression levels of phenotypic markers of osteoblastic differentiation in MC3T3‐E1 pre‐osteoblast. The purified peptide induced phosphorylation of mitogen‐activated protein kinases, including p38 mitogen‐activated protein kinase, extracellular regulated kinase, and c‐Jun N‐terminal kinase as well as Smads. As attested by molecular modelling study, the purified peptide interacted with the core interface residues in bone morphogenetic protein receptors with high affinity. Thus, the purified peptide could serve as a potential pharmacological substance for controlling bone metabolism.