Open-gate Conformation Research Articles

Open and Closed Structures of a Barium-Blocked Potassium Channel

Barium (Ba2+) is a classic permeant blocker of potassium (K+) channels. The “external lock-in effect” in barium block experiments, whereby the binding of external K+ impedes the forward translocation of the blocker, provides a powerful avenue to investigate the selectivity of the binding sites along the pore of potassium channels. Barium block experiments show that the external lock-in site is highly selective for K+ over Na+. Wild-type KcsA was crystallized in low K+ conditions, and the crystals were soaked in solutions containing various concentrations of barium. Structural analysis reveals open and closed gate conformations of the KcsA channel. Anomalous diffraction experiments show that Ba2+ primarily binds to the innermost site S4 of the selectivity filter of the open-gate conformation and also the site S2, but no binding is detected with the closed-gate conformation. Alchemical free-energy perturbation calculations indicate that the presence of a Ba2+ ion in the selectivity filter boosts the specificity of K+ binding relative to Na+ in the external sites S0–S2.

Free-Energy Landscapes of the Translocation of a Substrate in Four Proteasome - Activator Complexes Analyzed using Molecular Dynamics Simulations

Proteasome is involved in the degradation of proteins. Proteasome activators bind to the proteasome core particle (CP) and facilitate opening a gate of the CP, where Tyr8 and Asp9 in the N-termini tails of the CP form the ordered open gate. In a double mutant (Tyr8Gly/Asp9Gly), the N-termini tails are disordered and the stabilized open-gate conformation cannot be formed. To understand how proteasome is responsible for the efficient proteolysis of the substrate, four different molecular dynamics (MD) simulations were carried out: ordered- and disordered-gate models of the CP complexed with an ATP-independent PA26 and ordered- and disordered-gate models of the CP complexed with an ATP-dependent PAN-like activator. The free-energies of the translocation of a poly-peptide substrate moving through the gate were estimated using an MD simulation program called SCUBA developed by the Japan Atomic Energy Agency (JAEA). In the ordered-gate models, the substrate in the activator was more stable than that in the CP. The conformational entropy of the N-termini tails of the CP was larger when the substrate was in the activator than in the CP. In the disordered-gate models, the substrate in the activator was more destabilized than in the ordered-gate models. The mutated N-termini tails became randomized and their increased conformational entropy could no longer increase further even when the substrate was in the activator, meaning the randomized N-termini tails had lost the ability to stabilize the substrate in the activator. Thus, it was concluded that the dynamics of the N-termini tails entropically play a key role in the translocation of the substrate.

Essential function of the N-termini tails of the proteasome for the gating mechanism revealed by molecular dynamics simulations.

Proteasome is involved in the degradation of proteins. Proteasome activators bind to the proteasome core particle (CP) and facilitate opening a gate of the CP, where Tyr8 and Asp9 in the N-termini tails of the CP form the ordered open gate. In a double mutant (Tyr8Gly/Asp9Gly), the N-termini tails are disordered and the stabilized open-gate conformation cannot be formed. To understand the gating mechanism of the CP for the translocation of the substrate, four different molecular dynamics simulations were carried out: ordered- and Tyr8Gly/Asp9Gly disordered-gate models of the CP complexed with an ATP-independent PA26 and ordered- and disordered-gate models of the CP complexed with an ATP-dependent PAN-like activator. The free-energies of the translocation of a polypeptide substrate moving through the gate were estimated. In the ordered-gate models, the substrate in the activator was more stable than that in the CP. The conformational entropy of the N-termini tails of the CP was larger when the substrate was in the activator than in the CP. In the disordered-gate models, the substrate in the activator was more destabilized than in the ordered-gate models. The mutated N-termini tails became randomized and their increased conformational entropy could no longer increase further even when the substrate was in the activator, meaning the randomized N-termini tails had lost the ability to stabilize the substrate in the activator. Thus, it was concluded that the dynamics of the N-termini tails entropically play a key role in the translocation of the substrate.

Conformational Change in the ATPase-Gate of Topoisomerase VI Induced by Nucleotide and DNA

Archaeal topoisomerase VI catalyzes the relaxation and decatenation of DNA. In an ATP-dependent manner it transports a DNA double strand through a gap in a second duplex by a sequential opening and closing of two gates. Conformational change in the ATPase-gate of M. mazei topoisomerase VI was monitored in single molecule FRET experiments, using fluorescently labeled enzyme with one dye attached to each nucleotide-binding domain.We have observed four different conformational states of the ATP-binding domains: In the absence of nucleotide or DNA the ATPase-gate exhibits conformational flexibility, indicating gate opening and closing. Binding of supercoiled plasmid to TopoVI forces the ATPase-gate to open up even wider, although considerable flexibility of the domains is retained; both these states have been proposed on the basis of two crystal structures of intact TopoVI holoenzymes, showing a closed and an open gate conformation. Addition of ADPNP - a non-hydrolysable ATP analog - induces a well-defined conformation of the ATPase domains close to each other, indicating dimerization; this is in agreement with crystal structure from isolated TopoVI-B domains which show tight association when bound to ADPNP. Addition of both nucleotide and plasmid immobilizes the domains in a conformation different from the one observed for the closed gate, indicating that the presence of a DNA segment in the central cavity induces physical strain to the TopoVI complex; this state has not been proposed so far, but should be due to an internal rotation in the ATP-binding domains mediated by a conserved lysine residue acting as a gamma-phosphate sensor.Topoisomerase VI exhibits a different conformational behavior than type II topoisomerases (e.g. gyrase) in that it does not show DNA-induced pre-closure but rather opening of the gate in presence of a supercoiled DNA.

Modeling L-type Calcium Channel with Dihydropyridines

Dyhydropyridines (DHPs) is a major class of L-type calcium channel (LCC) ligands, which have boat-like six-membered ring with NH-group at the stern, aromatic moiety at the bow, and various substituents at the port and starboard sides. DHPs demonstrate antagonistic or agonistic action, which was previously explained as stabilization or destabilization, respectively, of the closed-gate state by the hydrophilic or hydrophobic port-side substituent. Here we used Monte Carlo energy-minimizations to dock various DHPs in the open-LCC model (Tikhonov & Zhorov, 2008). The calculations suggest a novel structural model in which agonistic and antagonistic actions are determined by different parts of the DHP molecule and have different molecular mechanisms. In our model, DHP polar moieties at the stern, bow, and starboard form H-bonds with side chains of Tyr_IVS6, Tyr_IIIS6, and Gln_IIIS5, respectively. The aromatic moiety at the bow binds to Phe_IIIP. We propose that these contacts with the well-known DHP-sensing residues stabilize the channel's open-gate conformation. Since these contacts are common for various DHPs, our model explains why both agonist and antagonists increase probability of the long-lasting channel openings and why even partial disruption of the contacts eliminates the agonistic action. In our model, the port-side is exposed to the permeation pathway and approaches the selectivity filter. Hydrophobic ports-side group of antagonists may induce long-lasting channel closings by destabilizing calcium coordination with the selectivity-filter glutamates in domains III and IV. In contrast, agonists, which have either hydrophilic or no substituent at the port-side, lack this destabilizing effect. Our model explains action of DHPs with diverse substituents. Thus, long substituents at the port-side are readily accommodated in the pore. Long substituents at the starboard-side protrude in the III/IV domain interface, explaining activity of DHPs linked to a permanently charged group. Supported by CIHR.

Blm10 binds to pre‐activated proteasome core particles with open gate conformation

Blm10 binds to pre‐activated proteasome core particles with open gate conformation

Blm10 binds to pre‐activated proteasome core particles with open gate conformation

Blm10 is bound to the yeast proteasome core particle, a crucial protease of eukaryotic cells [corrected]. Two gates, at both ends of the CP, control the access of protein substrates to the catalytic cavity of the CP. Normally, substrate access is auto-inhibited by a closed gate conformation unless regulatory complexes are bound to the CP and translocate protein substrates in an ATP-dependent manner. Here, we provide evidence that Blm10 recognizes pre-activated open gate CPs, which are assumed to exist in an equilibrium with inactive closed gate CP. Consequently, single-capped Blm10-CP shows peptide hydrolysis activity. Under conditions of disturbed CP assembly, as well as in open gate mutants, pre-activated CP or constitutively active CP, respectively, prevail. Then, Blm10 sequesters disordered and open gate CP by forming double-capped Blm10(2)-CP in which peptide hydrolysis activity is repressed. We conclude that Blm10 distinguishes between gate conformations and regulates the activation of CP.

Mechanism of Gate Opening in the 20S Proteasome by the Proteasomal ATPases

Substrates enter the cylindrical 20S proteasome through a gated channel that is regulated by the ATPases in the 19S regulatory particle in eukaryotes or the homologous PAN ATPase complex in archaea. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X (HbYX) motif that triggers gate opening upon ATP binding. Using cryo-electron microscopy, we identified the sites in the archaeal 20S where PAN's C-terminal residues bind and determined the structures of the gate in its closed and open forms. Peptides containing the HbYX motif bind to 20S in the pockets between neighboring alpha subunits where they interact with conserved residues required for gate opening. This interaction induces a rotation in the alpha subunits and displacement of a reverse-turn loop that stabilizes the open-gate conformation. This mechanism differs from that of PA26/28, which lacks the HbYX motif and does not cause alpha subunit rotation. These findings demonstrated how the ATPases' C termini function to facilitate substrate entry.

Investigation of the binding mode of (−)-meptazinol and bis-meptazinol derivatives on acetylcholinesterase using a molecular docking method

Molecular docking has been performed to investigate the binding mode of (-)-meptazinol (MEP) with acetylcholinesterase (AChE) and to screen bis-meptazinol (bis-MEP) derivatives for preferable synthetic candidates virtually. A reliable and practical docking method for investigation of AChE ligands was established by the comparison of two widely used docking programs, FlexX and GOLD. In our hands, we had more luck using GOLD than FlexX in reproducing the experimental poses of known ligands (RMSD<1.5 A). GOLD fitness values of known ligands were also in good agreement with their activities. In the present GOLD docking protocol, (-)-MEP seemed to bind with the enzyme catalytic site in an open-gate conformation through strong hydrophobic interactions and a hydrogen bond. Virtual screening of a potential candidate compound library suggested that the most promising 15 bis-MEP derivatives on the list were mainly derived from (-)-MEP with conformations of (S,S) and (SR,RS) and with a 2- to 7-carbon linkage. Although there are still no biological results to confirm the predictive power of this method, the current study could provide an alternate tool for structural optimization of (-)-MEP as new AChE inhibitors. [Figure: see text].

Nanogravimetric study of templated copper deposition in ion-channels of self-assembled glutathione films on gold piezoelectrodes

The ionic permeation and discharge processes at a controllable-gate ion-channel electrode were investigated. The ionically transmissible films were formed on Au piezoelectrodes by surface modification performed using self-assembled monolayers (SAM) of glutathione (GS) molecules adsorbed onto gold. The gating state of a AuSG film was controlled by utilizing uncomplexed Cu 2+ ions acting as the controller cations through their interactions with terminal carboxylic groups of AuSG, which results in a conformational transition leading to the film ion-channel opening. The open-gate conformation of AuSG is stabilized by lowering the solution pH. Under these conditions, we were able to record Cu 2+ ion transport and discharge at a Au substrate surface accessible through the open ion gates. We have found that despite of a very limited space of this access, in the first stage of the Cu 0 deposition process, a Cu-upd on Au is formed, with LSV peak potential E p = 0.385 V versus Ag/AgCl, though the rate of upd formation is lower than that for a upd on an unmodified Au, as evidenced by the decrease in the Cu deposited mass and the deposition charge. The value of the apparent charge transfer coefficient α app is also smaller ( α app = 0.36 compared to 0.52 for bare Au). The 3D nucleation and growth of copper nanocrystals was observed using the potential step i– t and m– t transients. The size of Cu 0 nuclei at the beginning of their overlapping process was in the range of d = 2–3 nm, which is larger than the ion-channel height ( h ≈ 1.22 nm) determined for the open-gate conformation of AuSG. We found no dynamic film instabilities associated with fast potential step programs.

Ion-gating phenomena of self-assembling glutathione films on gold piezoelectrodes

The adsorption of glutathione (GS) on a gold coated quartz crystal resonator surface was investigated to evaluate the saturation surface excess of GS, solvent content in the glutathione films on gold (AuSG), and conformational transitions in these films induced by interactions of AuSG with metal cations. It has been found that the resonant frequency shift recorded during the GS self-assembly agrees well with the resonator response predicted on the basis of molecular modeling of a GS film with GS molecules in the closed-gate conformation. The association interactions of AuSG film with Ba 2+ ions lead to a transformation of GS molecule to the open-gate conformation which is characterized by an elongated shape and results in solvent uptake by AuSG film. Other metal ions able to form carboxylate complexes, Pb 2+ and Ni 2+, were also found to be able to cause conformational transformation of GS molecule and open the ion gates in AuSG film. The film permeation by Pb 2+ ions is followed by Pb 0 deposition in the open channels and formation of nuclei at the film ∣ solution interface. In the case Ni 2+, the ion transport through the gates is followed by discharge and formation of a wedged interlayer between the Au substrate and the adsorbed GS molecules. Some organic compounds including neurotransmitters dopamine and serotonin and cationic drugs such as chlorpromazine, can also open the ion gates in AuSG films but conformational changes in the GS molecule are less reversible due to the partial retention of organic compounds in the film after medium exchange.

Developing an energy landscape for the novel function of a (beta/alpha)8 barrel: ammonia conduction through HisF.

HisH-hisF is a multidomain globular protein complex; hisH is a class I glutamine amidotransferase that hydrolyzes glutamine to form ammonia, and hisF is a (beta/alpha)8 barrel cyclase that completes the ring formation of imidizole glycerol phosphate synthase. Together, hisH and hisF form a glutamine amidotransferase that carries out the fifth step of the histidine biosynthetic pathway. Recently, it has been suggested that the (beta/alpha)8 barrel participates in a novel function: to channel ammonia from the active site of hisH to the active site of hisF. The present study presents a series of molecular dynamic simulations that investigate the channeling function of hisF. This article reconstructs potentials of mean force for the conduction of ammonia through the channel, and the entrance of ammonia through the strictly conserved channel gate, in both a closed and a hypothetical open conformation. The resulting energy landscape within the channel supports the idea that ammonia does indeed pass through the barrel, interacting with conserved hydrophilic residues along the way. The proposed open conformation, which involves an alternate rotamer state of one of the gate residues, presents only an approximately 2.5-kcal energy barrier to ammonia entry. Another alternate open-gate conformation, which may play a role in non-nitrogen-fixing organisms, is deduced through bioinformatics.