FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, R.
Right arrow Articles by Eckenhoff, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, R.
Right arrow Articles by Eckenhoff, R. G.
(The FASEB Journal. 2005;19:567-576.)
© 2005 FASEB

Structural basis for high-affinity volatile anesthetic binding in a natural 4-helix bundle protein

Renyu Liu, Patrick J. Loll* and Roderic G. Eckenhoff1

Department of Anesthesia, University of Pennsylvania, Philadelphia, Pennsylvania, USA; and
* Department of Biochemistry, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA

1Correspondence: Department of Anesthesia, 305 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6112, USA. E-mail: roderiic.eckenhoff{at}uphs.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Physiologic sites for inhaled anesthetics are presumed to be cavities within transmembrane 4-{alpha}-helix bundles of neurotransmitter receptors, but confirmation of binding and structural detail of such sites remains elusive. To provide such detail, we screened soluble proteins containing this structural motif, and found only one that exhibited evidence of strong anesthetic binding. Ferritin is a 24-mer of 4-{alpha}-helix bundles; both halothane and isoflurane bind with KA values of ~105 M–1, higher than any previously reported inhaled anesthetic-protein interaction. The crystal structures of the halothane/apoferritin and isoflurane/apoferritin complexes were determined at 1.75 Å resolution, revealing a common anesthetic binding pocket within an interhelical dimerization interface. The high affinity is explained by several weak polar contacts and an optimal host/guest packing relationship. Neither the acidic protons nor ether oxygen of the anesthetics contribute to the binding interaction. Compared with unliganded apoferritin, the anesthetic produced no detectable alteration of structure or B factors. The remarkably high affinity of the anesthetic/apoferritin complex implies greater selectivity of protein sites than previously thought, and suggests that direct protein actions may underlie effects at lower than surgical levels of anesthetic, including loss of awareness.—Liu, R., Loll, P. J. Eckenhoff, R. G. Structural basis for high-affinity volatile anesthetic binding in a natural 4-helix bundle protein.


Key Words: anesthesia • halothane • isoflurane • X-ray crystallography • calorimetry • ferritin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ACTIVITY of many membrane proteins in the central nervous system is altered by inhaled anesthetics (1) ; at least for injectable anesthetics, evidence indicates that ligand-gated ion channels make important contributions to the drug phenotype (2) . Delineation of a mechanism has been hampered by a lack of structural detail for these complex receptors and any evidence for direct anesthetic binding. Nevertheless, available data suggest that important binding regions lie within membrane-embedded helical bundles. For example, photolabeling has identified a state-sensitive volatile anesthetic binding site in the transmembrane region of the nicotinic acetylcholine channel (3) . The acetylcholine receptor is the structural prototype for the entire ligand-gated ion channel superfamily, and a recent model based on cryoelectron microscopy data allows placement of this site in the core of a membrane 4-{alpha}-helix bundle. That this site is functionally relevant is suggested by electrophysiology and transgenic animal experiments in which mutagenesis of residues in the analogous region of the GABAA receptor altered the anesthetic effects (2 , 4 , 5) . Although this implicates the transmembrane regions of receptors or ion channels in the binding of volatile anesthetics, binding does not necessarily require the hydrophobic membrane environment, since designed 4-{alpha}-helix bundles are also able to bind volatile anesthetics within their hydrophobic cores (6) . However, even these simple 4-helix bundle systems have so far resisted attempts at detailed structural characterization. This lack of a tractable model system for studying anesthetic binding prompted us to test a series of natural proteins containing 4-helix bundles for the ability to bind volatile anesthetics. Anesthetic binding to this series was not common, but the search led to the discovery that ferritin exhibits unusually strong binding for volatile anesthetics.

Ferritin is a large oligomeric protein, functioning at a minimum to concentrate iron for subsequent release when required for synthesis of heme and other iron cofactors (7) ; the coupled role is to maintain cellular free iron concentrations at a nontoxic level. Each 20 kDa ferritin monomer adopts a 4-helix bundle fold. Monomers associate to form 2-fold symmetric dimers; 12 dimers associate into a 24-mer having 432 symmetry. The 24-mer forms a spherical shell enclosing an enormous central cavity that can hold >4000 iron atoms.

Preexisting cavities or packing defects in the hydrophobic interiors of proteins are thought to be the preferred binding sites for volatile anesthetic ligands (8 9 10) . No such cavities exist within the ferritin 4-helix bundle light chain (L) monomer, as indicated by surface calculations using CASTp (PDB ID 1IER, http://cast.engr.uic.edu/cast/) (11) . However, cavities of suitable volume and shape are observed at the LL dimer interfaces in the ferritin oligomer, at the position of the 2-fold symmetry axes. This suggests that 12 binding sites for volatile anesthetics such as halothane should exist on the intact ferritin molecule. We present experimental evidence confirming the existence of such binding sites, which reveals that these sites possess remarkably high affinity for the anesthetic. In addition, we present the underlying structural basis of anesthetic binding, as elucidated by X-ray diffraction analysis, of the ferritin-anesthetic complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Halothane and isoflurane were obtained from Halocarbon Laboratories (River Edge, NJ, USA); racemic preparations of both anesthetics were used in the crystallization experiments. Dichlorohexafluorocyclobutane (F6) was obtained from PCR Chemical (Gainesville, FL, USA). Apoferritin and ferritin were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and used without further purification. Apoferritin was used for most studies since X-ray diffraction is not possible with iron-loaded ferritin. All other chemicals were obtained from Sigma and were of reagent grade.

Isothermal titration calorimetry
Titrations were performed at 20°C using a Microcal, Inc. VP ITC (http://www.microcalorimetry.com/). The ITC consists of a matched pair of sample and reference vessels (1.43 mL) enclosed in an adiabatic enclosure and a rotating stirrer/syringe for titrating aliquots of the ligand solution into the sample vessel. The sample vessel contained 25 µM apoferritin in 130 mM NaCl, 20 mM NaHPO4, pH 7.0, and the reference vessel contained water. Ligand (1.5 mM for halothane and isoflurane) was titrated into the protein sample cell, using volumes of 10 µL for the first five injections and 20 µL thereafter, with 5 min intervals between injections. Sequential titrations were performed until the titration signal was essentially constant. Final anesthetic concentration in the cell was ~0.5 mM. Sequential titrations were linked using ConCat32 software (Microcal, Inc., Northampton, MA, USA), then analyzed with Origin 5.0 (Microcal, Inc.), making corrections for heats due to ligand into buffer, buffer into protein, and buffer into buffer. The model used to fit the ITC data are based on the equation, Q = MtV0(n1{Theta}1{Delta}H1 + n2{Theta}2{Delta}H2), where Q is the heat content after any injection; Mt is the total concentration of macromolecule in the active cell volume, Vo; n1 and n2 are the numbers of binding sites for set 1 and set 2, {Theta} 1 and {Theta} 2 are the fraction of sites occupied by the ligand for set 1 and set 2, and {Delta}H1 and {Delta}H2 are the molar heats of ligand binding for set 1 and set 2, respectively. {Theta} is related to its respective K and the free concentration of ligand [X] by the equations K = {Theta}/{(1– {Theta})[X]}, where [X] = Xt – Mt(n1{Theta}1 + n2{Theta}2) and Xt is the total concentration of ligand. Note that Mt, Xt, and V0 are known. Parameters n, {Delta}H, and K for each set are adjusted to minimize the sum of squares of differences between the measured heats and those predicted from this model, using the Marquardt method. Once K and {Delta}H are determined, {Delta}S can be calculated using the standard formulas {Delta}G = {Delta}H – T{Delta}S = –RTlnK.

ITC competition experiments
To test whether halothane and isoflurane can compete with one another for ferritin binding, 17 µM apoferritin was placed in the sample cell and titrated with 1.5 mM solutions of the first anesthetic, followed by a second titration with a 1.5 mM solution of the other anesthetic. As a control, initial titrations were carried out with buffer only, followed by a second titration with 1.5 mM halothane or isoflurane.

Crystallization and complex formation
Large single crystals were grown in hanging drops at 4°C in 1 wk, using a reservoir solution of 0.05 M cadmium sulfate, 0.1 M HEPES, and 1.0 M sodium acetate, pH 7.5. A protein solution containing 12 mg/mL apoferritin in 0.03 M NaCl was mixed 1:1 with reservoir buffer; crystals grew to full size in ~ 1 wk. For anesthetic complexes, an identical crystallization procedure was followed, except that the droplet was suspended over 2 mL glass vials sealed with vacuum grease and the reservoir solution used was saturated with halothane or isoflurane. Before data collection, crystals were cryoprotected in reservoir buffer containing 30% sucrose saturated with halothane or isoflurane and flash-cooled in liquid nitrogen.

Data collection and processing
Diffraction data were collected from anesthetic complex crystals maintained at 100 K at beamline X8C of the National Synchrotron Light Source. For the halothane data set, the wavelength was set to the wavelength corresponding to the peak of the bromine K edge. Diffraction data from a native crystal (i.e., a crystal never exposed to anesthetic) were collected at a home X-ray source using Cu K{alpha} radiation from a microfocus rotating anode source. Details of the data collection are given in Table 1 .


View this table:
[in this window]
[in a new window]
  		Table 1. Diffraction data statisticsa

The ferritin crystals used for diffraction grew in space group F432 and were isomorphous with previously determined ferritin crystal structures (12 , 13) . Protein coordinates from PDB file 1GWG were used as a starting model for the refinements. Initial models containing protein atoms only were used for automated water placement and refinement using Arp-Warp and Refmac (14 , 15) . Cadmium ions present in the lattice were identified as peaks in the anomalous difference density maps. Ligands were placed into difference maps using XtalView (16) ; refinement of the complex structure was completed was using Refmac. Coordinates for halothane were obtained from Tang et al. (17) and for isoflurane from the Dundee PRODRG server (http://davapc1.bioch.dundee.ac.uk/programs/prodrg/) (18) . Coordinates for the halothane/apoferritin and isoflurane/apoferritin complexes have been deposited in the Protein Data Bank (accession nos. 1XZ1 and 1XZ3, respectively).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isothermal titration calorimetry
The interaction of halothane and isoflurane with apoferritin yielded sigmoidal enthalpograms (Fig. 1 , upper panels). The calculated Wiseman "c" parameter ranged between 1 and 6, lying within the experimental k window (1-100) wherein parameters can be fitted reliably (19) . The stoichiometry of binding (n), association constant (KA), enthalpy change {Delta}H, and entropy change ({Delta}S) were derived from fits to one- and two-site models. The two-class binding site model resulted in a 5-fold reduction in chi-square values (Fig. 1 , lower panels; Table 2 ), and predicted ~ four high-affinity sites and eight lower affinity sites, in agreement with the total number of sites predicted from the structure of the apoferritin oligomer. The holoprotein ferritin also bound anesthetics with similar parameters (data not shown).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 1. Upper panels: Isothermal titration of apoferritin with halothane (left) and isoflurane (right) showing exothermic nature. Initial 5 injections each used 10 µL of 10 mM halothane and subsequent injections added 20 µL of the same solution. Lower panels: Enthalpograms and the 2-class binding site model fits (see Table 2 for parameters and text for details of the fitting). Single class binding site fits resulted in similar overall parameters (n~8, K~20,000 M–1, {Delta}H ~ –5 kcal/mol, {Delta}S ~3 cal/mol/°K), but the chi-square values were ~ 5-fold higher.


View this table:
[in this window]
[in a new window]
  		Table 2. Thermodynamic parameters from ITC data

The anesthetic/apoferritin interaction was found to have favorable enthalpic and entropic contributions. Isoflurane resulted in greater heat release than halothane, but calculated KA values were similar. Titration of apoferritin with isoflurane completely inhibited the heat release of a subsequent halothane titration (Fig. 2 ). On the other hand, titration of apoferritin with halothane did not fully inhibit the heat release of a subsequent isoflurane titration, indicating that isoflurane occupies the same sites as halothane but with a somewhat larger enthalpy change.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Competition ITC experiments. Presaturation of apoferritin with isoflurane resulted in no further heat release on titration with halothane (left); presaturation with halothane resulted in significant further heat release on isoflurane titration (right).

Dichlorohexafluorocyclobutane (F6)
This compound was included as a control in the calorimetry experiments. F6 has similar structural and physicochemical character as halothane and isoflurane but lacks anesthetic effects at predicted concentrations (20 , 21) . As with anesthetics, the interaction with apoferritin was exothermic, but the enthalpogram revealed distinctly different ITC parameters. Best fits of the data revealed 1.1 ± 0.04 sites with a KA of 5.6 ± 0.5 x 104 M–1, indicating that the majority of sites in apoferritin are selective for an anesthetic compound rather than for a comparably sized, nonanesthetic hydrophobic molecule.

Anesthetic complex structure determination
The anesthetic binding cavity lies between two apoferritin monomers, directly on the 2-fold axis of symmetry relating these two monomers (Fig. 3 ). This 2-fold symmetry axis, which is inherent in the structure of the ferritin oligomer, coincides with a crystallographic 2-fold axis. Therefore, any ligand binding within this cavity will exhibit 2-fold disorder—i.e., two positions will be seen for the ligand, related by a 180° rotation, with each position having a maximum occupancy of 50%. This disorder necessarily complicates the structure determination; fortunately, however, the high resolution of the data aided interpretation of the electron density in the binding site.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 3. Left: Backbone representation of the apoferritin 24-mer, with helices represented by cylinders. Two monomers related by 2-fold symmetry (dark and light) are highlighted in the black box. Right: Expanded view of the highlighted dimer with a bound isoflurane molecule shown in a space-filling representation.

In the case of the halothane ligand, the availability of anomalous differences arising from the presence of a bromine atom in the ligand allowed the position and orientation of the anesthetic molecule to be unambiguously determined. The only peaks in the halothane anomalous difference map (apart from peaks associated with known Cd2+ positions) were found in the anesthetic binding site and were readily modeled as the S- and R-isomers of halothane and their 2-fold related positions (Fig. 3) . A racemic mixture of S- and R-halothane was used to prepare the complex; however, from the relative integrated intensities of the peaks in the anomalous difference map, the S-isomer was seen to predominate, with the S/R ratio being estimated at ~2:1.

A racemic mixture of isoflurane was also used, but it was not possible to unambiguously identify which enantiomer of the anesthetic is bound. S-isoflurane was used in the refinement. Because the ITC data suggested full occupancy of sites under the crystal conditions, occupancies were set to 0.5 for halothane and isoflurane symmetrical pairs and were not refined; the reasonable values for the individual atomic B-values of the anesthetics obtained during the refinement suggest that these occupancy estimates are not far from correct.

Halothane/apoferritin
Halothane is found in a small hydrophobic cavity lying at the interface between two apoferritin monomers (Fig. 3) . This cavity has two mouths, which open to the interior of the apoferritin oligomer (Fig. 4 ). The cavity has a solvent accessible surface area of 288 Å2 and volume of 142 Å3, as calculated using a 1.4 Å radius probe in CASTp. If one alters the probe radius to a size approximating the short axis of halothane (2.9 Å), the calculated solvent-accessible volume of this cavity is reduced to ~0.1Å3, demonstrating that the ligand fits snugly in at least two dimensions.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 4. Electrostatic surface renderings of the anesthetic binding pocket. The bar at left shows the color coding of the electrostatic surface; red is acidic, blue is basic. a) The ferritin dimer viewed from inside the apoferritin cavity. The 2-fold symmetry axis relating the two apoferritin monomers that comprise the dimer is perpendicular to plane of the page and passes through the center of the dimmer, where 2 mouths can be seen opening into the anesthetic binding cavity, each surrounded by a region of basic charge (openings indicated by yellow arrows. b) Same orientation as panel a, but expanded and with a clipping plane positioned to allow the viewer to see the interior of the anesthetic binding cavity. One of the 2 symmetry-related halothane ligands is shown in a ball-and-stick representation. The 2 symmetry-related copies of Tyr-28 are shown in a space-filling representation; these 2 side chains effectively define 2 opposite sides of the cavity. c) Anesthetic binding cavity rotated 90° about a horizontal axis with respect to orientation of panel b. The electrostatic surface is now shown as a semitransparent mesh surface. The vantage point is from within the interior of the apoferritin molecule, looking into the cavity, where 1 halothane ligand may be seen. In this orientation, one can clearly see the channels that lead outward from the cavity to the protein surface; the basic mouths of these channels are marked with blue arrows.

No single obvious strong polar interaction between protein and ligand can be identified from the structure. However, there are several potential weak polar interactions (Table 3 and Fig. 5 ). For example, fluorine atoms of S-halothane, which are predicted to carry substantially negative partial charges (17) , lie ~ 4 Å from the hydroxyl oxygen of Ser-27 and may be engaging in a hydrogen bond-like electrostatic interaction (22) . The fluorine atoms are seen to point toward the carbonyl carbon of Ser-27 and the C{alpha} proton of Tyr-28, and hence occupy what has been described as a favorable environment for organic fluorine atoms bound to proteins (23) . Bromine and chlorine atoms of halothane are predicted to carry a slight negative charge (24 , 25) . In S-halothane, the bromine lies 3.1 Å from the carbonyl oxygen of Leu-24 and 4 Å from its hydrogen-bonded partner, nitrogen of Tyr-28, perhaps indicating an interaction with the positive hydrogen. The halothane chlorine atom lies above the edge of the Tyr-28 ring, approaching to within 3.3 Å of a ring carbon atom. The positions of the bromine and chlorine are reversed in the R-enantiomer, allowing the chlorine to interact with the Leu-24/Tyr-28 hydrogen bond and the bromine to pack against the edge of the Y28 aromatic ring. B-values for the halothane atoms are between 45 and 50 Å2, roughly twice the average protein atom B-value.


View this table:
[in this window]
[in a new window]
  		Table 3. Anesthetic-protein atomic distances



View larger version (56K):
[in this window]
[in a new window]
  		Figure 5. Anesthetic-protein interactions. a) Stereo view of halothane in the anesthetic binding site. Different colored backbone ribbons represent the two symmetry-related apoferritin monomers. Oxygen, red; nitrogen, blue; fluorine, purple; bromine, orange; chlorine, green. The halothane ligand is shown with black bonds; both of the 2-fold related halothane positions are indicated. Note that 1 of the 4 helices making up the binding site is partially cut away to reveal the ligand (lower yellow helix, in foreground). b) Stereo view of isoflurane in the anesthetic binding site. For clarity’s sake, only 1 of the 2 symmetry-related isoflurane molecules is shown. Color scheme same as in panel a.

No global differences in protein structure could be detected upon comparison of the unliganded apoferritin structure with that of the halothane complex. After superposition of the two structures, the RMS deviation in C{alpha} positions is 0.10 Å, less than or equal to the expected level of error for the final refined model. The RMS deviation in position for all atoms is larger, 0.51 Å; however, all large differences map to poorly ordered surface side chains far from the anesthetic binding site. The variation of atomic B-values with residue number is essentially identical for the unliganded protein and the halothane complex, indicating that no gross changes in backbone mobility accompany anesthetic binding. CASTp analysis indicated that changes in cavity volume and surface area are small between the liganded and unliganded structures, with the solvent-accessible volume and area (using 1.4 Å radius probe) being 142 Å3 and 288 Å2, respectively, in the unliganded structure and 141 Å3 and 273 Å2 in the halothane-occupied structure.

Isoflurane/apoferritin
Isoflurane binds in the same position as halothane (Fig. 5b ). The cavity volume and surface area are similar to those of the halothane and unliganded structures. Once again, multiple weak interactions instead of s few strong ones were noted. The chlorine (which in isoflurane is predicted to carry a partial negative charge; S. Vemparala and M. L. Klein, University of Pennsylvania, unpublished results) in the modeled enantiomer lies 3.6 Å from the hydroxyl oxygen of Ser-27; one of the difluoromethyl fluorines on the 2-fold related copy of the ligand lies 3.5 Å from the same serine hydroxyl (Table 3) . Both of these distances are considerably less than the sum of van der Waals distances, assuming there is an intervening hydrogen atom. The difluoromethyl fluorine atoms are in close proximity to the carbonyl group of Ser-27 and the C{alpha} proton of Tyr-28, as was seen for the fluorine atoms in halothane. A trifluoromethyl fluorine atom is within 3.2 Å of the aromatic ring of Tyr-28; the fluorine atom approaches the edge of the ring rather than the ring face, as would be expected for an electrostatic interaction between the partially negative fluorine atom and a partially positive tyrosine ring hydrogen. The closest atom to the ether oxygen is the hydroxyl oxygen of Ser-27, but the distance of 4.6 Å suggests this is not energetically significant. As for the halothane/apoferritin structure, there are no significant differences in protein conformation or B-values between the isoflurane complex and the unliganded protein. B-values for the isoflurane atoms lie between 47 and 53 Å2, again roughly twice the average B-value for the protein atoms.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Inhaled anesthetics bind specifically to many proteins and modulate their activities (1 , 10 , 26) , but there have been few examples where these interactions have been characterized structurally. Firefly luciferase is known to be inhibited by general anesthetics and the structure of an anesthetic/luciferase complex has been determined (27) , but the protein has only been crystallized in the ATP-free, low-affinity state and with a nonclinical anesthetic, bromoform. The 2.4 Å structure of the halothane/human serum albumin complex has also been reported (28) . In luciferase and serum albumin, the anesthetic occupies preexisting hydrophobic cavities that serve as binding sites for other, nonanesthetic ligands; in both cases the anesthetic-protein interaction appears to be very weak (KA<500 M–1), (29 , 30) being driven largely by the hydrophobic effect (30) .

Preexisting cavities are also important for anesthetic binding to apoferritin. However, the affinity of the interaction between apoferritin and either halothane or isoflurane is at least 100-fold stronger than that seen with luciferase and serum albumin. The calorimetry data show that this high affinity is achieved with a combination of entropic and enthalpic contributions. The former likely arise from desolvation of the anesthetic (hydrophobic effect) whereas the latter arise from more specific interactions within the binding site as demonstrated in this study. Indeed, the number of favorable polar interactions and potential hydrogen bonds in the anesthetic/apoferritin complex exceeds that in the HSA/halothane complex by ~ 2-fold (Table 3) , and more polar residues are found in the apoferritin cavity lining (Table 4 ).


View this table:
[in this window]
[in a new window]
  		Table 4. Residues contributing to pocket surface

Anesthetic-protein interactions
The steric fit of the anesthetic in the apoferritin pocket reflects favorable van der Waals packing interactions, even though the ligand/cavity packing density is not high. The ratio of ligand volume (approximated by a 2.9 Å radius sphere) to the molecular surface volume defined by that sphere (CASTp) in the apoferritin cavity is 0.55. This value for the packing density has been suggested to be optimal for host-guest interactions (31) ; higher packing densities are opposed by entropic losses. The packing density for halothane in the HSA domain III anesthetic binding site is almost 0.8, suggesting that the anesthetic’s 100-fold lower affinity for HSA is due to entropic costs. Indeed, the binding of halothane with HSA domain III is characterized by a negative {Delta}S (unpublished results), whereas in the apoferritin/halothane interaction {Delta}S is positive. Nevertheless, the considerable enthalpic component in the case of apoferritin suggests that electrostatic interactions within the pocket are making important contributions. These interactions are relatively long range, and therefore weak. Cumulatively, however, and particularly in the low dielectric environment of this cavity, these interactions are the most likely contributors to the immobilization and selective orientation of the anesthetic. Three residues that appear to contribute most of the favorable interactions are tyrosine 28, serine 27, and leucine 24. The halogen atoms of the ligands make weak electrostatic contacts to atoms from these residues in a manner analogous to haloalkane dehalogenase, where the chlorines of dichloroethane form electrostatic contacts with the ring hydrogens of tryptophan and phenylalanine (32) . Similar polar interactions between fluorine atoms of a ligand and polar protein side chains have also been observed in molecular dynamics simulations of the halothane/gramicidin interaction (22) . Only a single such interaction is found in the HSA/halothane complex (28) , where a trifluoromethyl fluorine approaches to within 3.2 Å of a ring carbon of Phe-403; no doubt the lack of polar interactions between anesthetic and HSA contributes to the lower affinity. In addition to the polar interactions described above, the proximity of bromine (halothane) or chlorine (halothane or isoflurane) atoms with different polar protein atoms may produce favorable van der Waals interactions in apoferritin’s anesthetic binding pocket. It is notable that the relatively acidic hydrogens of halothane and isoflurane do not appear to participate in hydrogen bonds with any protein atom in the pocket.

The bromine and chlorine atoms of halothane and one of the fluorine atoms of isoflurane make a close approach to the Tyr-28/Leu-24 N-O hydrogen bond. It has been proposed (33) that anesthetics might interact with and disrupt backbone hydrogen-bonded networks, but in the case of apoferritin anesthetic binding this potential interaction has no significant effect on the length (and presumably the strength) of this bond.

Selectivity
Isoflurane has a higher enthalpic contribution to binding, but an overall similar affinity as halothane, because of a less favorable entropic term. This is consistent with the larger volume of isoflurane and the necessarily increased packing density (~0.7). We had initially presumed that the enhanced enthalpy of the isoflurane/apoferritin interaction relative to that with halothane would be due to an interaction with the ether oxygen. However, our data do not show any specific interactions between the protein and the ether oxygen, but suggest instead that the longer isoflurane molecule allows for a closer approach of the fluorine atoms to Ser-27 and Tyr-28.

The crystallographic data indicate that the S-enantiomer of halothane is preferred over the R-form, perhaps due to the small differences in partial charge between the chlorine and bromine atoms that allow for improved interactions between the protein and the S-enantiomer. The only in vivo evidence of halothane enantiomeric selectivity is found in a mutant of the nematode C. elegans, where the R-enantiomer was somewhat more potent than the S (34) . There is mammalian evidence for enhanced potency of R-isoflurane over S- (~20%), but it was impossible to unambiguously determine the stereochemical configuration of bound ligand(s) in the isoflurane structure. Because the in vivo enantioselectivity is a likely result of an ensemble of molecular selectivities, there is no a priori reason to expect a similar magnitude or direction in apoferritin.

Heterogeneous binding sites
ITC data suggest at least two classes of binding site exist in the intact 24-mer. The most likely explanation is the heterogeneous nature of horse spleen apoferritin. This preparation consists of ~15% heavy chains (H-ferritin) and 85% light chains (L-ferritin) (7) . Because the anesthetic binding sites in the ferritin oligomer lie at the interface between two monomers, it is expected that the presence of different isoforms will lead to different classes of binding sites. After LL, the most probable dimer is HL, which should correspond to ~30% of the possible dimers, or 4 per intact ferritin 24-mer. Since this estimate matches the number of high-affinity sites in the optimal enthalpogram fit, we propose that the HL dimer includes a higher affinity binding site than LL, either at its interface or in the H-monomer itself. Unfortunately, our conditions favored crystallization of the all-L form, so this hypothesis cannot be directly addressed using the commercially available mixture of apoferritin isomers. Analyzing the human H ferritin structure (93% identical to horse, PDB ID# 2FHA) with CASTp, we find the HH interfacial cavity to be smaller and the interhelical (monomeric) cavity larger. The monomeric H cavity is of interest because it is hydrophobic, aromatic-rich, and contains the ferroxidase site.

Another potential explanation for the existence of binding sites with different affinities is heterogeneity in the Arg-59 conformation. Arg-59 adopts two conformers in the ferritin structure, which we have labeled A and B. Since the anesthetic binding site lies on a 2-fold symmetry axis, two copies of the Arg-59 side chain lie near the drug, and there are four possible combinations of Arg-59 conformers in each binding site—AA', AB', BA', and BB'—where the prime denotes which of the two symmetry-related protein molecules contributes the arginine side chain (note that AB' and BA' are equivalent). Switching between these conformers has a great effect on the cavity size and shape. Thus, it is possible that Arg-59 can partially control access and affinity of the anesthetic binding site. For example, the high-affinity sites might be expected to have a packing density approaching the "ideal" 55%, which for both anesthetics appears to be the AA' form. The lower affinity set of sites may reflect a larger cavity volume, lower packing density (~16%), and a loss of van der Waals interactions, such as are seen in the BB' form.

Apoferritin as a model
The fold of the apoferritin monomer is a 4-{alpha}-helix bundle analogous to the transmembrane region of the Cys-loop superfamily of ligand-gated ion channels (35) , which have long been considered to contribute to inhaled anesthetic action (36) . Mutagenesis of specific subunits from the GABAA inhibitory subtype of ion channels has implicated residues within these transmembrane bundles as controlling anesthetic sensitivity (2 , 4) . Halothane photolabeling experiments with the nicotinic acetylcholine receptor have shown the existence of a state-dependent binding site in the same general area (3) , which recent cryoelectron microscopy studies predict is in the core of the transmembrane 4-helix bundle (35) . The anesthetic binding site in apoferritin is located at the interface between two 4-{alpha}-helix bundles instead of within the core of a single monomer. Nevertheless, the "fold" of this interface remains that of an antiparallel 4-{alpha}-helix bundle. The anesthetic is sandwiched between tyrosine, serine, and apolar residues in an arrangement that optimizes interactions with the halogens. At least in the case of the nicotinic acetylcholine receptor {delta} subunit based on the 4Å model, the photolabeled site is similar—a pocket is formed between two tyrosine residues (Y228 and Y291), several apolar residues (I295, L278, L292) and finally a hydroxyl (T281, instead of a serine). The analogous GABAA site remains ambiguous because of the lack of structure, but mutation of potential electrostatic partners in the same region, a serine ({alpha}2-S270) (37) or an asparagine (ß3-N265), altered the anesthetic sensitivity (2) .

The interfacial, dimeric nature of this binding site may be of some importance, since movements of one monomer with respect to the other might allow additional conformational flexibility and allow an optimal induced fit of ligands. This may be the basis for high-affinity (200 µM) anesthetic binding in a designed dimeric 4-helix bundle (6) , since a monomeric 3-helix bundle with a similarly designed cavity provided only very weak anesthetic binding (38) . The analogous interfacial domain in the ligand-gated channel superfamily is the subunit-subunit interface in the transmembrane region. If anesthetic binding sites similar to those seen in ferritin exist at these intersubunit interfaces, anesthetics could modulate the oligomerization equilibria that must underlie assembly and perhaps function of these receptor/channels. Alternatively, occupancy of the central transmembrane core implicated by photolabeling and mutagenesis may modulate the helix motion that underlies gating.

Apoferritin as a target
The role of this interfacial cavity in ferritin’s normal function is not clear, but S27, Y28, and R59 are highly conserved residues (7) . Other ligands noted to bind in this cavity are betaine ((carboxymethyl)trimethylammonium ion) (39) , and porphyrin (40) , but the energetics of these complexes have not been reported. It is of interest that such a high-affinity inhaled anesthetic site still allows this degree of degeneracy, reminiscent of sites found in human serum albumin (28 , 30) . Internal cavities generally allow rearrangements of atoms at lower energetic cost than if the protein matrix was well packed, and thus these cavities may facilitate conformational transitions. Occupancy of these cavities should improve packing and stability, and raise the energetic barrier to changes in conformation. However, such an effect is likely to be small given the already high stability of ferritin and the fact that we could detect no changes in B-factors in the anesthetic complex. Alternatively, if the hypothesis regarding preferential binding to the HL dimer interface is validated, anesthetics would be expected to modulate the stoichiometry of ferritin assembly, which, depending on cell or organ needs, could have important physiologic effects.

Disruption in iron metabolism, movement or concentrations may have important cellular consequences. For example, iron is a key catalyst in the Fenton reaction, capable of enhancing free-radical mediated cytotoxicity under specific circumstances (41) . Because of these effects, iron metabolism has been implicated as an important proximal contributor in a host of neurodegenerative disorders (42) . Alteration in iron movement and availability by the anesthetic might therefore trigger or enhance neuronal toxicity. It is relevant to note here that anesthetic-induced apoptosis and neurodegeneration has been observed in the developing brain (43) , and postoperative cognitive dysfunction is now well documented after middle age (44 , 45) . Thus, while it seems unlikely that ferritin is a target underlying anesthesia, it is clearly a high-affinity target that might underlie a spectrum of side effects. It is intriguing that side effect targets may be of higher affinity than primary effect targets.

In conclusion, the apoferritin/inhaled anesthetic complex represents the highest affinity interaction for those clinically important compounds described. The structural basis for this affinity is a combination of an optimal host-guest fit and multiple weak electrostatic contacts and weak hydrogen bonds, instead of a single strong interaction. The acidic hydrogen, and the ether oxygen appear not to make energetically significant contributions to this complex. The existence of such high-affinity sites of potential importance for understanding structure activity relationships provides credibility to the notion that direct protein actions may underlie behavioral effects at lower than surgical concentrations of anesthetic, including loss of awareness.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 6.



View larger version (9K):
[in this window]
[in a new window]
  		Figure 7.


   ACKNOWLEDGMENTS
 
The authors appreciate the initial structural assistance of Steven Stayrook, Ph.D., the insight into ferritin structure and function from Ivan Dmochowski, Ph.D., the biochemical assistance of Jin Xi, M.S., and Q.C. Meng, Ph.D., and the graphical assistance of Brad Jameson. Data for this study were measured at beamline X8C of the National Synchrotron Light Source. Financial support for the NSLS comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the National Institutes of Health. Financial support from this work was provided by NIGMS P-01 55876 (R.G.E.) and the Fanny Ripple Foundation (P.J.L.).

Received for publication September 20, 2004. Accepted for publication December 2, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Campagna, J. A., Miller, K. W., Forman, S. A. (2003) Mechanisms of actions of inhaled anesthetics. N. Engl. J. Med. 348,2110-2124[Free Full Text]
  2. Jurd, R., Arras, M., Lambert, S., Drexler, B., Siegwart, R., Crestani, F., Zaugg, M., Vogt, K. E., Ledermann, B., Antkowiak, B., et al (2003) General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J. 17,250-252[Abstract/Free Full Text]
  3. Chiara, D. C., Dangott, L. J., Eckenhoff, R. G., Cohen, J. B. (2003) Identification of nicotinic acetylcholine receptor amino acids photolabeled by the volatile anesthetic halothane. Biochemistry 42,13457-13467[CrossRef][Medline]
  4. Mihic, S. J., Ye, Q., Wick, M. J., Koltchine, V. V., Krasowski, M. D., Finn, S. E., Mascia, M. P., Valenzuela, C. F., Hanson, K. K., Greenblatt, E. P., et al (1997) Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature (London) 389,385-389[CrossRef][Medline]
  5. Trudell, J. (2002) Unique assignment of inter-subunit association in GABA(A) alpha 1 beta 3 gamma 2 receptors determined by molecular modeling. Biochim. Biophys. Acta 1565,91-96[Medline]
  6. Johansson, J. S., Scharf, D., Davies, L. A., Reddy, K. S., Eckenhoff, R. G. (2000) A designed four-alpha-helix bundle that binds the volatile general anesthetic halothane with high affinity. Biophys. J. 78,982-993[Abstract/Free Full Text]
  7. Theil, E. C. (2001) Ferritin. Messerschmidt, A. Huber, R. Poulos, T. Wieghardt, K. eds. Handbook of Metalloproteins ,771-781 John Wiley & Sons, Ltd. Chichester.
  8. Johansson, J. S., Manderson, G. A., Ramoni, R., Grolli, S., Eckenhoff, R. G. (2005) Binding of the volatile general anesthetics halothane and isoflurane to a mammalian beta-barrel protein. FEBS J. 272,573-581
  9. Eckenhoff, R. G. (2001) Promiscuous ligands and attractive cavities: how do the inhaled anesthetics work?. Mol. Interv. 1,258-268[Abstract/Free Full Text]
  10. Xi, J., Liu, R., Asbury, G. R., Eckenhoff, M. F., Eckenhoff, R. G. (2004) Inhalational anesthetic-binding proteins in rat neuronal membranes. J. Biol. Chem. 279,19628-19633[Abstract/Free Full Text]
  11. Liang, J., Edelsbrunner, H., Woodward, C. (1998) Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design. Protein Sci. 7,1884-1897[Abstract]
  12. Hempstead, P. D., Yewdall, S. J., Fernie, A. R., Lawson, D. M., Artymiuk, P. J., Rice, D. W., Ford, G. C., Harrison, P. M. (1997) Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution. J. Mol. Biol. 268,424-448[CrossRef][Medline]
  13. Evans, G., Bricogne, G. (2002) Triiodide derivatization and combinatorial counter-ion replacement: two methods for enhancing phasing signal using laboratory Cu Kalpha X-ray equipment. Acta Crystallogr. D Biol. Crystallogr. D58,976-991
  14. Lamzin, V. S., Perrakis, A., Wilson, K. S. (2001) The ARP/WARP suite for automated construction and refinement of protein models. Rossmann, M. G. Arnold, E. eds. International Tables for Crystallography ,720-722 Dordrecht, Kluwer Academic Publishers The Netherlands.
  15. Murshudov, G. N., Vagin, A. A., Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. D53,240-255
  16. McRee, D. E. (1992) XtalView: A VISUAL PROTEIN CRYSTALLOGRAPHIC SOFTWARE SYSTEM for XII/XView. J. Mol. Graph. 10,44-47[CrossRef]
  17. Tang, P., Zubryzcki, I., Xu, Y. (2001) Ab Initio calculation of structures and properties of halogenated general anesthetics: halothane and sevoflurane. J. Comp. Chem. 22,436-444[CrossRef]
  18. Schuttelkopf, A. W., Van Aalten, D. M. (2004) PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60,1355-1363[CrossRef][Medline]
  19. Wiseman, T., Williston, S., Brandts, J. F., Lin, L. N. (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179,131-137[CrossRef][Medline]
  20. Kendig, J. J., Kodde, A., Gibbs, L. M., Ionescu, P., Eger, E. I. (1994) Correlates of anesthetic properties in isolated spinal cord: cyclobutanes. Eur. J. Pharm. 264,427-436[CrossRef][Medline]
  21. Raines, D. E. (1996) Anesthetic and nonanesthetic halogenated volatile compounds have dissimilar activities on nicotinic acetylcholine receptor desensitization kinetics. Anesthesiology 84,663-671[CrossRef][Medline]
  22. Tang, P., Xu, Y. (2002) Large-scale molecular dynamics simulations of general anesthetic effects on the ion channel in the fully hydrated membrane: the implication of molecular mechanisms of general anesthesia. Proc. Natl. Acad. Sci. USA 99,16035-16040[Abstract/Free Full Text]
  23. Olsen, J. A., Banner, D. W., Seiler, P., Wagner, B., Tschopp, T., Obst-Sander, U., Kansy, M., Muller, K., Diederich, F. (2004) Fluorine interactions at the thrombin active site: protein backbone fragments H-C(alpha)-C=O comprise a favorable C-F environment and interactions of C-F with electrophiles. ChemBioChem 5,666-675[CrossRef][Medline]
  24. Scharf, D., Laasonen, K. (1996) Structure, effective pair potential and properties of halothane. Chem. Phys. Lett. 258,276-282[CrossRef]
  25. Liu, Z., Xu, Y., Saladino, A. C., Wymore, T., Tang, P. (2004) Parametrization of 2-bromo-2-chloro-1,1,1-trifluoroethane (halothane) and hexafluoroethane for nonbonded interactions. J. Phys. Chem. A 108,786-792
  26. Eckenhoff, M. F., Eckenhoff, R. G. (1998) Quantitative autoradiography of halothane binding in rat brain. J. Pharmacol. Exp. Ther. 285,371-376[Abstract/Free Full Text]
  27. Franks, N. P., Jenkins, A., Conti, E., Lieb, W. R., Brick, P. (1998) Structural basis for the inhibition of firefly luciferase by a general anesthetic. Biophys. J. 75,2205-2211[Abstract/Free Full Text]
  28. Bhattacharya, A. A., Curry, S., Franks, N. P. (2000) Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures. J. Biol. Chem. 275,38731-38738[Abstract/Free Full Text]
  29. Dickinson, R., Franks, N. P., Lieb, W. R. (1993) Thermodynamics of anesthetic/protein interactions. Temperature studies on firefly luciferase.. Biophys. J. 64,1264-1271[Abstract/Free Full Text]
  30. Liu, R., Pidikiti, R., Ha, C. E., Petersen, C. E., Bhagavan, N. V., Eckenhoff, R. G. (2002) The role of electrostatic interactions in human serum albumin binding and stabilization by halothane. J. Biol. Chem. 277,36373-36379[Abstract/Free Full Text]
  31. Mecozzi, S., Rebek, J., Jr (1998) The 55% solution: a formula for molecular recognition in the liquid state. Chem. Eur. J. 4,1016-1022[CrossRef]
  32. Verschueren, K. H., Seljee, F., Rozeboom, H. J., Kalk, K. H., Dijkstra, B. W. (1993) Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase. Nature (London) 363,693-698[CrossRef][Medline]
  33. Di Paulo, T., Sandorfy, C. (1974) Fluorocarbon anaesthetics break hydrogen bonds. Nature (London) 252,471-472[CrossRef][Medline]
  34. Sedensky, M. M., Meneely, P. M. (1987) Genetic analysis of halothane sensitivity in Caenorhabditis elegans. Science 236,952-954[Abstract/Free Full Text]
  35. Miyazawa, A., Fujiyoshi, Y., Unwin, N. (2003) Structure and gating mechanism of the acetylcholine receptor pore. Nature (London) 423,949-955[CrossRef][Medline]
  36. Franks, N. P., Lieb, W. R. (1994) Molecular and cellular mechanisms of general anaesthesia. Nature (London) 367,607-614[CrossRef][Medline]
  37. Mascia, M. P., Trudell, J. R., Harris, R. A. (2000) Specific binding sites for alcohols and anesthetics on ligand-gated ion channels. Proc. Natl. Acad. Sci. USA 97,9305-9310[Abstract/Free Full Text]
  38. Manderson, G. A., Johansson, J. S. (2002) Role of aromatic side chains in the binding of volatile general anesthetics to a four-alpha-helix bundle. Biochemistry 41,4080-4087[CrossRef][Medline]
  39. Trikha, J., Theil, E. C., Allewell, N. M. (1995) High resolution crystal structures of amphibian red-cell L ferritin: potential roles for structural plasticity and solvation in function. J. Mol. Biol. 248,949-967[CrossRef][Medline]
  40. Precigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C., Langlois d’Estaintot, B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D50,739-743
  41. Halliwell, B. (1992) Reactive oxygen species and the central nervous system. J. Neurochem. 59,1609-1623[Medline]
  42. Ke, Y., Ming, Q. Z. (2003) Iron misregulation in the brain: a primary cause of neurodegenerative disorders. Lancet Neurol. 2,246-253[CrossRef][Medline]
  43. Jevtovic-Todorovic, V., Hartman, R. E., Izumi, Y., Benshoff, N. D., Dikranian, K., Zorumski, C. F., Olney, J. W., Wozniak, D. F. (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J. Neurosci. 23,876-882[Abstract/Free Full Text]
  44. Moller, J. T., Cluitmans, P., Rasmussen, L. S., Houx, P., Rasmussen, H., Canet, J., Rabbitt, P., Jolles, J., Larsen, K., Hanning, C. D., et al (1998) Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet 351,857-861(erratum: Lancet, 1998, vol. 351, p. 1742)[CrossRef][Medline]
  45. Johnson, T., Monk, T., Rasmussen, L. S., Abildstrom, H., Houx, P., Korttila, K., Kuipers, H. M., Hanning, C. D., Siersma, V. D., Kristensen, D., et al (2002) Postoperative cognitive dysfunction in middle-aged patients. Anesthesiology 96,1351-1357[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Anesth. Analg.Home page
R. G. Eckenhoff
Why Can All of Biology Be Anesthetized?
Anesth. Analg., September 1, 2008; 107(3): 859 - 861.
[Full Text] [PDF]

Home page
 Anesth. Analg.Home page
M. B. Kelz and R. G. Eckenhoff
Does It Add Up?
Anesth. Analg., August 1, 2008; 107(2): 365 - 366.
[Full Text] [PDF]

Home page
 Biophys. JHome page
J. H. Streiff, T. W. Allen, E. Atanasova, N. Juranic, S. Macura, A. R. Penheiter, and K. A. Jones
Prediction of Volatile Anesthetic Binding Sites in Proteins
Biophys. J., November 1, 2006; 91(9): 3405 - 3414.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
S. Vemparala, L. Saiz, R. G. Eckenhoff, and M. L. Klein
Partitioning of Anesthetics into a Lipid Bilayer and their Interaction with Membrane-Bound Peptide Bundles
Biophys. J., October 15, 2006; 91(8): 2815 - 2825.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, R.
Right arrow Articles by Eckenhoff, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, R.
Right arrow Articles by Eckenhoff, R. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS