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November 2013

November 2013

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Mayo Foundation for Medical Education and Research

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SUPPLEMENTARY DATA

Halothane Binds to Druggable Sites in [Ca2+]4-CaM, but Does Not

Inhibit [Ca2+]4-CaM Activation of Kinase

NENAD O. JURANIĆ1*, KEITH A. JONES2, ALAN R. PENHEITER1, THOMAS J.

HOCK2, and JOHN H. STREIFF2

1 Mayo College of Medicine at Rochester, U.S.A. and 2University of Alabama at

Birmingham, U.S.A..

NMR TITRATION DATA

Backbone amide 1H-15N peaks positions determined from 3D HNCO titration spectra

Titration curves for the prominent residues from each CaM lobe. Red arrow indicates

mole ratio at which NMR structure determination of CaM/halothane complex is done.

ORIGINAL VERSION OF FIGURE 3 FROM THE PAPER

Figure 3. Models of five conformers for N-terminus (to the left, 2KUG.pdb) and C-

terminus (to the right, 2KUH.pdb) lobes of [Ca2+]4-CaM –halothane complex. Halothane

is given in red color, protein in green. The atomic stick-plot is overlaid with protein

cartoon (helices are denoted by letters).

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Halothane binds to druggable sites in the [Ca2+]4-calmodulin (CaM) complex, but does not inhibit [Ca2+]4-CaM activation of kinase

Article

January 2013

Nenad Juranic · Keith A. Jones · Alan R. Penheiter · Thomas J. Hock · John H. Streiff

Article

Full-text available

Halothane binds to druggable sites in the [Ca2+]4-calmodulin (CaM) complex, but does not inhibit [Ca...

January 2013 · Journal of the Serbian Chemical Society

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Computational methods designed to predict and visualize ligand protein binding interactions were used to characterize volatile anesthetic (VA) binding sites and unoccupied pockets within the known structures of VAs bound to serum albumin, luciferase, and apoferritin. We found that both the number of protein atoms and methyl hydrogen, which are within approximately 8 A of a potential ligand ... [Show full abstract] binding site, are significantly greater in protein pockets where VAs bind. This computational approach was applied to structures of calmodulin (CaM), which have not been determined in complex with a VA. It predicted that VAs bind to [Ca(2+)](4)-CaM, but not to apo-CaM, which we confirmed with isothermal titration calorimetry. The VA binding sites predicted for the structures of [Ca(2+)](4)-CaM are located in hydrophobic pockets that form when the Ca(2+) binding sites in CaM are saturated. The binding of VAs to these hydrophobic pockets is supported by evidence that halothane predominantly makes contact with aliphatic resonances in [Ca(2+)](4)-CaM (nuclear Overhauser effect) and increases the Ca(2+) affinity of CaM (fluorescence spectroscopy). Our computational analysis and experiments indicate that binding of VA to proteins is consistent with the hydrophobic effect and the Meyer-Overton rule.

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The main objective of this work was to characterize VA binding sites in multiple anesthetic target proteins. A computational algorithm was used to quantify the solvent exclusion and aliphatic character of amphiphilic pockets in the structures of VA binding proteins. VA binding sites in the protein structures were defined as the pockets with solvent exclusion and aliphatic character that exceeded ... [Show full abstract] minimum values observed in the VA binding sites of serum albumin, firefly luciferase, and apoferritin. We found that the structures of VA binding proteins are enriched in these pockets and that the predicted binding sites were consistent with experimental determined binding locations in several proteins. Autodock3 was used to dock the simulated molecules of 1,1,1,2,2-pentafluoroethane, difluoromethyl 1,1,1,2-tetrafluoroethyl ether, and sevoflurane and the isomers of halothane and isoflurane into these potential binding sites. We found that the binding of the various VA molecules to the amphiphilic pockets is driven primarily by VDW interactions and to a lesser extent by weak hydrogen bonding and electrostatic interactions. In addition, the trend in Delta G binding values follows the Meyer-Overton rule. These results suggest that VA potencies are related to the VDW interactions between the VA ligand and protein target. It is likely that VA bind to sites with a high degree of solvent exclusion and aliphatic character because aliphatic residues provide favorable VDW contacts and weak hydrogen bond donors. Water molecules occupying these sites maintain pocket integrity, associate with the VA ligand, and diminish the unfavorable solvation enthalpy of the VA. Water molecules displaced into the bulk by the VA ligand may provide an additional favorable enthalpic contribution to VA binding. Anesthesia is a component of many health related procedures, the outcomes of which could be improved with a better understanding of the molecular targets and mechanisms of anesthetic action.