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- Bertaccini, Edward J, et al.
(författare)
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Modeling Anesthetic Binding Sites within the Glycine Alpha One Receptor Based on Prokaryotic Ion Channel Templates : The Problem with TM4
- 2010
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Ingår i: Journal of chemical information and modeling. - : American Chemical Society (ACS). - 1549-960X .- 1549-9596. ; 50:12, s. 2248-2255
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Tidskriftsartikel (refereegranskat)abstract
- Ligand-gated ion channels (LGICs) significantly modulate anesthetic effects. Their exact molecular structure remains unknown. This has led to ambiguity regarding the proper amino acid alignment within their 3D structure and, in turn, the location of any anesthetic binding sites. Current controversies suggest that such a site could be located in either an intra- or intersubunit locale within the transmembrane domain of the protein. Here, we built a model of the glycine alpha one receptor (GlyRa1) based on the open-state structures of two new high-resolution ion channel templates from the prokaryote, Gloebacter violaceus (GLIC). Sequence scoring suggests reasonable homology between GlyRa1 and GLIC. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3): (ILE229, SER 267, and ALA 288). They line an intersubunit interface, in contrast to previous models. However, residues from the fourth transmembrane domain (TM4) that are known to modulate a variety of anesthetic effects are quite distant from this putative anesthetic binding site. While this model can account for a large proportion of the physicochemical data regarding such proteins, it cannot readily account for the alterations on anesthetic effects that are due to mutations within TM4.
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| 2. |
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| 3. |
- Bertaccini, Edward J, et al.
(författare)
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Toxin Binding Serves as an Initial Model for Studying the Effects of Anesthetics on Ion Channels
- 2007
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Konferensbidrag (refereegranskat)abstract
- Introduction: We have previously used molecular modeling techniques combined with experimental data to visualize a plausible model of an anesthetic binding site within a LGIC complex.1 We have also previously shown a computational mechanism by which these ion channels may open and close and postulated how this motion may be affected by the presence of anesthetics.2 The difficulties with these methods, however, lay in their inability to account for the modest effects of a separate anesthetic ligand or small mutation on ion channel motion. Here we show the successful application of an elastic network calculation on a homologue of the extracellular component of LGIC's, the acetycholine binding protein (AChBP), in the presence and absence of large cobratoxin ligands. These calculations demonstrate a clear alteration in the native symmetric motion of a protein due to the presence of multiple ligands, as may occur with anesthetics and muscle relaxants. Methods: Coordinates of the AChBP with (1YI5)3 and without (1I9B)4 cobratoxin were obtained from the Research Collaboratory for Structural Biology (RCSB). Hydrogens were added using DSViewer 5.0 (Accelrys, San Diego, CA). Normal mode analysis was performed using an all atom elastic network model developed by Lindahl. Root-mean-square deviations (RMSD) of each residue were produced from the application of the RMSD analysis utility within the GROMACS software suite to the coordinate trajectory output files. The RMSD data was then imported into Microsoft Excel for plotting and further comparison of protein backbone motions between the two different normal mode trajectories. Results: Normal mode analysis reveals that ligand binding to this protein alters its natural harmonic vibration. In this case, the axially symmetric motion of the AChBP, that may be associated with channel gating in the full nAChR, is highly dampened by the presence of bound cobratoxin. A large proportion of the kinetic energy within this mode seems to be absorbed by the cobratoxin, leaving the channel motion significantly decreased. Conclusions: This is among the first descriptions of the effect of bound ligand on large scale protein dynamics, especially as it relates to ion channel gating. This analysis was possible using an elastic network approximation due to the large protein nature of the cobratoxin ligand. For nonpeptide drugs such as anesthetics which contain far fewer atoms, using the effects of bound ligand on protein motion as additional criteria for future drug design may require a more robust molecular mechanics treatment of the ligand-receptor complex.
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| 4. |
- Bertaccini, Edward J., et al.
(författare)
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Anesthetic Binding Sites in a GlyRa1 Model Based on Open State Prokaryotic Ion Channel Templates
- 2009
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Ingår i: Proceedings of the 2009 Annual Meeting of the American Society Anesthesiologists.
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Konferensbidrag (refereegranskat)abstract
- Introduction : Ligand-gated ion channels (LGICs) are thought to mediate a significant proportion of anesthetic effects. We built atomic level models of the glycine alpha one receptor (GlyRa1) to examine its interactions with anesthetics. We previously built models of a GlyRa1 based on a prokaryotic pentameric ion channel in the closed state from Erwinia Chrysanthemi (ELIC) (1-3). Here, we built a GlyRa1 model based on the open state structures of two new ion channels from the prokaryote Gloebacter violaceus (GLIC).(4-5) These new templates are relevant since anesthetics are thought to bind to and stabilize the open state of the GlyRa1. Methods : The 3D coordinates of two forms of GLIC (3EHZ.pdb and 3EAM.pdb) were obtained from the RCSB database. The sequence of the human GlyRa1 was obtained from the NCBI database. A BLAST sequence search was performed using the GLIC sequences. Among the best scored homologous human sequences were those of the GlyRa1. The template structures and the sequence of GlyRa1 were aligned with Discovery Studio 2.0.1 (Accelrys, San Diego, CA) and the Modeler module was used for assignment of coordinates for aligned amino acids, the construction of possible loops, and the initial refinement of amino acid sidechains. Results : The BLAST derived scores suggest a close homology between the LGICs, GLIC and ELIC. Subsequent CLUSTALW alignment of the GLIC and GlyRa1 sequences demonstrates reasonable sequence similarity. The model of the GlyRa1 is a homomer with pentameric symmetry about a central ion pore and shows significant transmembrane alpha helical and extracellular beta sheet content. Unlike our previous model based on the ELIC template, the current model based on the GLIC templates shows a continuously open pore with a partial restriction within the transmembrane region. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3) (ILE229, SER 267, ALA 288). They now line the intersubunit interface, in contrast to our previous models. However, residues from TM4 that are known to modulate a variety of anesthetic effects on this or homologous LGICs are present but could only indirectly influence an intersubunit anesthetic binding site. Normal mode analyses show an iris-like motion similar to previous results.Conclusions : A model of the GlyRa1 was constructed using homology modeling based on the GLIC templates. This model posits an intersubunit site for anesthetic binding that may communicate with the intrasubunit region of each TMD.
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| 5. |
- Bertaccini, Edward J., et al.
(författare)
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Normal-mode analysis of the glycine alpha1 receptor by three separate methods
- 2007
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 47:4, s. 1572-1579
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Tidskriftsartikel (refereegranskat)abstract
- Predicting collective dynamics and structural changes in biological macromolecules is pivotal toward a better understanding of many biological processes. Limitations due to large system sizes and inaccessible time scales have prompted the development of alternative techniques for the calculation of such motions. In this work, we present the results of a normal-mode analysis technique based on molecular mechanics that enables the calculation of accurate force-field based vibrations of extremely large molecules and compare it with two elastic network approximate models. When applied to the glycine alpha1 receptor, all three normal-mode analysis algorithms demonstrate an "iris-like" gating motion. Such gating motions have implications for understanding the effects of anesthetic and other ligand binding sites and for the means of transducing agonist binding into ion channel opening. Unlike the more approximate methods, molecular mechanics based analyses can also reveal approximate vibrational frequencies. Such analyses may someday allow the use of protein dynamics elucidated via normal-mode calculations as additional endpoints for future drug design.
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| 6. |
- Bertaccini, Edward J, et al.
(författare)
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Understanding Effects of Anesthetics on Ligand-Gated Ion Channels (LGIC) in Lipid Membranes
- 2008
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Konferensbidrag (refereegranskat)abstract
- Introduction: We have previously used molecular modeling combined with experimental data to visualize a plausible model of an anesthetic binding site within a LGIC.1 We have also previously shown a computational mechanism by which these LGICs may gate and postulated how this motion may be affected by the presence of anesthetics.2 The initial difficulty with these calculations concerns the 26000 atoms present in the receptor and the computing capabilities required to perform vibrational analyses on such a large construct. Here we show the successful application of an elastic network calculation on our previously published model of a glycine alpha one receptor (GlyRa1), now suspended in a fully hydrated lipid bilayer. Despite the presence of over 100,000 atoms , these calculations continue to demonstrate a symmetric motion of the ion channel protein that is consistent with the gating motion demonstrated in previous in vacuo work by us and others. Methods: Coordinates of the GlyRa1 model were obtained from our previous work. A 100x100A lipid bilayer matrix was constructed from POPC and then hydrated on both surfaces with water molecules using the VMD 1.86 software package (NCSA, Urbana, Ill.). Discovery Studio 1.7 (Accelrys, San Diego, CA) molecular modeling software was used to insert our GlyRa1 model into the lipid bilayer such that the known interfacial residue GLY 221 was at the POPC-water interface. All waters within 3.8A of the protein were removed as were all lipid molecules within 2A of the protein. Hydrogens were added followed by energy minimization of the entire system to remove energetically unfavorable contacts. The system was subsequently further hydrated within the GROMACS software suite and subjected to further energy equilibration via molecular dynamics simulation with periodic boundary conditions. Subsequent normal mode analysis was performed using an all atom elastic network model developed by Lindahl which takes advantage of a sparse matrix implementation for computational efficiency. Results: Despite the large size of the system, the introduction of water and lipid did not grossly distort the overall gating motion of the glyRa1 noted in previous works. Normal mode analysis revealed that the GlyRa1 in a fully hydrated bilayer environment continues to demonstrate an iris-like gating motion as a low frequency, high amplitude natural harmonic vibration. Furthermore, the introduction of periodic boundary conditions allowed simultaneous harmonic vibrations of lipid in sync with the protein gating motion that are compatible with reasonable lipid bilayer perturbations. Conclusions: This is among the first description of a normal mode calculation describing large-scale protein dynamics and ion channel gating in the presence of a fully hydrated lipid bilayer complex. This analysis was only possible on such a large system due to the computational efficiencies of the elastic network approximation. This model will hopefully provide a more accurate means of introducing anesthetics and alcohols into protein and lipid bilayer systems and allow us to discern their effects on LGIC gating. 1Bertaccini EJ, Shapiro J, Brutlag DL, Trudell JR: J Chem Inf Model 2005; 45: 128-35; 2Bertaccini EJ, Trudell JR, Lindahl E:J Chem Inf Model 2007; 47: 1572-9.
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| 9. |
- Yoluk, Özge, et al.
(författare)
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Stabilization of the GluCl Ligand-Gated Ion Channel in the Presence and Absence of Ivermectin
- 2013
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 105:3, s. 640-647
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Tidskriftsartikel (refereegranskat)abstract
- Improving our understanding of the mechanisms and effects of anesthetics is a critically important part of neuroscience. The currently dominant theory is that anesthetics and similar molecules act by binding to Cys-loop receptors in the postsynaptic terminal of nerve cells and potentiate or inhibit their function. Although structures for some of the most important mammalian channels have still not been determined, a number of important results have been derived from work on homologous cationic channels in bacteria. However, partly due to the lack of a nervous system in bacteria, there are a number of questions about how these results relate to higher organisms. The recent determination of a structure of the eukaryotic chloride channel, GluCl, is an important step toward accurate modeling of mammalian channels, because it is more similar in function to human Cys-loop receptors such as GABA(A)R or GlyR. One potential issue with using GluCl to model other receptors is the presence of the large ligand ivermectin (IVM) positioned between all five subunits. Here, we have performed a series of microsecond molecular simulations to study how the dynamics and structure of GluCl change in the presence versus absence of IVM. When the ligand is removed, subunits move at least 2 angstrom closer to each other compared to simulations with IVM bound. In addition, the pore radius shrinks to 1.2 angstrom, all of which appears to support a model where IVM binding between subunits stabilizes an open state, and that the relaxed nonIVM conformations might be suitable for modeling other channels. Interestingly, the presence of IVM also has an effect on the structure of the important loop C located at the neurotransmitter-binding pocket, which might help shed light on its partial agonist behavior.
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