Multiple Binding Sites for the General Anesthetic Isoflurane Identified in the Nicotinic Acetylcholine Receptor Transmembrane Domain

Multiple binding sites for the general anesthetic isoflurane identified in the nicotinic acetylcholine receptor transmembrane domain

Grace Brannigana,1, David N. LeBarda, Jérôme Héninb, Roderic G. Eckenhoffc, and Michael L. Kleina,1

aInstitute for Computational and Molecular Science, Temple University, Philadelphia, PA 19122; bLaboratoire d’Ingénierie des Systèmes Macromoléculaires, Centre National de la Recherche Scientifique—Aix-Marseille Université, 13402 Marseille, France; and cDepartment of Anesthesiology and Critical Care, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Contributed by Michael L. Klein, June 24, 2010 (sent for review May 12, 2010)

An extensive search for isoflurane binding sites in the nicotinic An intriguing difference between the nAChR structure acetylcholine receptor (nAChR) and the proton gated ion channel reported in 2BG9 and the prokaryotic structures are the large from Gloebacter violaceus (GLIC) has been carried out based on mo- gaps in protein density in the extracellular half of the nAChR lecular dynamics (MD) simulations in fully hydrated lipid membrane transmembrane domain (TMD). The high-resolution prokaryotic environments. Isoflurane introduced into the aqueous phase readily structures do not display such gaps (Fig. S1). Recently (14), we partitions into the lipid membrane and the membrane-bound pro- proposed that such gaps are occupied by cholesterol, which is tein. Specifically, isoflurane binds persistently to three classes of essential for nAChR function (15). Because cholesterol is not sites in the nAChR transmembrane domain: (i) An isoflurane dimer found in prokaryotic membranes, this hypothesis provides an occludes the pore, contacting residues identified by previous muta- alternate explanation to the source of differences between the genesis studies; analogous behavior is observed in GLIC. (ii) Several two structures. Furthermore, as we demonstrate in the present nAChR subunit interfaces are also occupied, in a site suggested paper, even with docked cholesterol, there is ample space in by photoaffinity labeling and thought to positively modulate the the nAChR TMD for binding of multiple isoflurane molecules. iii receptor; these sites are not occupied in GLIC. ( ) Isoflurane binds The collapse of the nAChRTMD observed in simulations of a cho- BIOPHYSICS AND to the subunit centers of both nAChR α chains and one of the GLIC lesterol-free model (14) results in a structure that presumably COMPUTATIONAL BIOLOGY chains, in a site that has had little experimental targeting. Inter- would not offer as many binding sites for anesthetics as preted in the context of existing structural and physiological data, observed here. Such a collapse, however, is inconsistent with struc- the present MD results support a multisite model for the mechanism tural information obtained on nAChR in native membranes (13), of receptor-channel modulation by anesthetics. and consequently no cholesterol-free nAChR models were considered in this study. anesthesia ∣ cys-loop receptor ∣ ligand-gated ion channel In the absence of detailed structural information, most experimental efforts to determine binding sites for anesthetics in espite efforts reaching back over a century, the molecular Cys-loop receptors have depended on techniques such as electro- Dmechanism through which certain small molecules (general physiology, mutagenesis, and photolabeling of various anesthetics anesthetics) cause reversible immobilization and amnesia re- and alcohols to the GABAA, glycine, and nACh receptors. At mains unclear. Known general anesthetics fall into several diverse clinical concentrations, most volatile anesthetics positively modu- classes, but the dominant effects of nearly all general anesthetics late the GABAA receptor but negatively modulate the nAChR, are believed to reflect modulation of ion channels in the central indicating that some binding sites do not overlap (16). In general, nervous system (1–3). An understanding of the mechanisms by experiments suggest multiple binding sites (17, 18) for anesthetics which general anesthetics modulate such channels is therefore not only essential for medical progress, but can also serve to and alcohols on both the nAChR and GABAA receptor: Potential sites have been identified in the TMD, at subunit interfaces (1, 3, illuminate underlying behavior of ion channels and their larger – – role in the biological processes of mobilization and conscious- 16, 19 24) in the nAChR pore, (16, 25 29), and at various ness. Particular attention has focused on the anesthetic-sensitive positions in the agonist-binding domain (22). The multitude of Cys-loop superfamily of ligand-gated ion channels, including potential sites and mechanisms has particularly complicated inter- cation channels such as the nicotinic acetylcholine receptor pretation of ion current measurements, because of the possibility (nAChR) and serotonin receptors, as well as anion channels such of competing effects. Mutagenesis and photolabeling studies as the γ-aminobutyric acid class A (GABAA) receptor and the provide an incomplete picture of anesthetic binding sites, because glycine receptor. Recently a prokaryotic cation channel of this the choice of mutations or selective reactivity of the photolabel superfamily, the proton gated ion channel from Gloebacter viola- prevent the whole receptor from being explored, and the hydro- ceus (GLIC), has demonstrated sensitivity to both intravenous phobic regions to which anesthetics bind are difficult to isolate. and inhaled anesthetics at subclinical concentrations (4). In addition, such methods typically identify regions of the amino High-resolution crystal structures have demonstrated that acid sequence, from which spatial location of binding sites is indir- anesthetics do bind directly to proteins (5–8), whereas more ectly inferred. If multiple residues are identified, it is often not indirect means such as mutagenesis and photolabeling have indicated that general anesthetics bind to Cys-loop receptors – Author contributions: G.B., D.N.L., J.H., R.G.E., and M.L.K. designed research; G.B. and in particular (1 3). Obtaining high-resolution structures of Cys- D.N.L. performed research; G.B. and D.N.L. analyzed data; and G.B., D.N.L., J.H., R.G.E., loop receptors even in the absence of anesthetic has proven to be and M.L.K. wrote the paper. a challenge, however, and structures have only been solved for The authors declare no conflict of interest. – prokaryotic pentameric ion channels (9 11), including GLIC in Freely available online through the PNAS open access option. a putatively open state (3EHZ, 3EAM). For the most part, these 1To whom correspondence may be addressed. E-mail: [email protected] or gbrannigan@ crystal structures reveal a family of proteins that is consistent with temple.edu. Torpedo the earlier 4-Å cryo-EM structure of nAChR from solved This article contains supporting information online at www.pnas.org/lookup/suppl/ by Unwin and coworkers (2BG9) (12, 13). doi:10.1073/pnas.1008534107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008534107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 clear whether they form multiple binding sites, or simply represent water, which is shown in ref. 8 to be an essential component of multiple sides of the same binding site. some anesthetic binding sites. The membrane lipid environment Complementing experimental approaches, computational can also be included, along with the cholesterol that we proposed methods can serve to directly illuminate microscopic features of (14) is embedded in the nAChR TM-gaps. Furthermore, the MD anesthetic-ion channel interactions. Structure-based docking is approach accounts for interactions between anesthetic molecules, a common technique used to find ligand binding sites on a protein and can therefore identify multiply occupied sites. of known structure. Tang and coworkers (30) reported several Flooding of a protein by anesthetics has been reported pre- mostly superficial sites for halothane detected using structure- viously by members of our group (31, 32); here we present based docking to their model of the α4β2 nAChR in an open MD simulations on a much increased scale involving isoflurane conformation; binding free energy calculations revealed that ha- partitioning into two nearly complete and fully solvated penta- lothane bound with low affinity to most sites, with the exception of meric ligand-gated ion channels: nAChR and GLIC. In order a deeper TM site suggested by experiments. Molecular dynamics to achieve full partitioning of isoflurane into deeply buried (MD) computation-based “flooding” of the receptor (in which a protein sites, this method requires substantially longer computa- high concentration of anesthetic is placed in the surrounding water tion times than those reached by previous simulations of Cys-loop and allowed to partition into lipid and protein binding sites over receptors. Such simulations typically involve about 200,000 the course of an MD simulation trajectory) is a more expensive atoms. The two systems presented here were simulated for alternative to structure-based docking which holds several advan- 0.4 μs each. The nAChR from Torpedo was used for the simula- tages for investigating volatile anesthetics and Cys-loop receptors. tions for the most direct comparison with experimental data and In the MD approach, nearly all protein degrees of freedom are to reduce errors caused by homology modeling. unrestrained, resulting in a fully flexible, dynamic, and physical We find that isoflurane binding is remarkably consistent dock that is especially appropriate for the highly mobile nAChR between GLIC and nAChR, with the exception of sites deep with- (and the corresponding medium-resolution structure). Unlike in the TMD. We divide the binding sites we observe in the docking calculations, the MD approach naturally includes explicit nAChR into eight classes, four of which are found in the TMD

Fig. 1. Regions of persistent occupation by isoflurane. (A and B) Isoflurane binding sites in the nAChR. Protein is colored by subunit: α, blue; β, purple; δ, green; γ, cyan. Embedded cholesterol is yellow. Colored blobs represent an isoflurane density isosurface averaged over the last 100 ns of the simulation; large blobs represent occupation over at least most of that period, whereas a few much smaller blobs represent occupation for less than half of that period. (A) Side view of the αδ, γ, and αγ subunits, as well as isoflurane sites contacting those subunits. Isoflurane binding sites in the TMD are colored as follows: superficial/annular sites (gray), intrasubunit sites (red), intersubunit sites (orange), and pore site (brown). Isoflurane binding sites in the LBD are pink, blue, and yellow, corresponding with binding to the agonist site, beta sandwiches, and α1 helices, respectively. (B) View of the nAchR TM domain, looking down on the membrane from the extracellular region. (C and D) Isoflurane binding sites in GLIC. Protein is green and blobs are colored as in A–C, with the addition of isoflurane in the loop site (purple). (C) Side view of three chains of GLIC. (D) TMD of GLIC, with isoflurane in loop site, intrasubunit site, pore site, and annular sites. Binding to LBD for both proteins is shown in Figs. S4–S6

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1008534107 Brannigan et al. Downloaded by guest on September 27, 2021 (pore, intersubunit, intrasubunit, and annular), and four of which A are found in the extracellular domain (interfacial loops, agonist site, β-sandwich, and α1 helix). Binding to internal sites in the TMD (intersubunit and intrasubunit sites) is much reduced in the tightly packed GLIC structure, whereas other classes of sites are similarly occupied in the two receptors. In Results, we address the three sites with the strongest implications for channel function, and remaining sites are addressed in the SI Text. The data presented here only provide direct information regarding the location of binding sites, and not their effect on the receptor B 0.25 structure and dynamics. Furthermore, the present paper reports nAChR only on the location of sites of at least moderate affinity as 0.2 demonstrated by persistent occupancy. Quantitative measure- 0.15 GLIC

ments of affinity that reliably include even relatively fast protein (z)

dynamics (such as rotation of side-chain dihedral angles) would iso require significantly more computational time, as demonstrated P 0.1 in ref. 33. 0.05 Results By the final frames of the simulation, there were between zero 0 and one isoflurane molecules in the aqueous phase surrounding 6’ 10’ 13’ 16’ 20’ the nAChR, corresponding to a concentration ranging between 0 z and 1 mM; this range includes the EC50 for anesthesia as well as Fig. 2. Distribution of isoflurane density in ion channel pore. (A) Standard the concentrations used in most experiments cited here. Iso- prime numbering scheme for both nAChR (2) and GLIC (11). Figure is based on flurane surrounding the GLIC simulation equilibrated to a con- that of ref. 2. (B) Probability to find at least one isoflurane molecule with centration of 4 mM. As demonstrated in Fig. S2, fewer isoflurane height z in the pore. Both curves include combined data from each isoflurane molecules partitioned to the GLIC surface than the nAChR molecule in the dimer.

surface. BIOPHYSICS AND Binding sites were identified by constructing an isoflurane threonine residues found in nAChR. Although there is presently COMPUTATIONAL BIOLOGY density map, averaged over frames from the last 100 ns of the no experimental data regarding the location of binding sites for simulation. Regions of high density therefore reflect sites in anesthetics on GLIC, binding of anesthetics to the pore provides which isoflurane was persistently bound, and are shown for both the simplest explanation for the inhibitory effect reported in ref. 4. nAChR and GLIC in Fig. 1. A few sites (depicted as small blobs) The results of several experiments with differing methodolo- were occupied for less than half of the production period. Contact gies are consistent with a binding site for anesthetics in the residues in the TMD are reported in Fig. S3. nAChR pore. In addition to the mutagenesis studies (28, 29) already mentioned, photolabeling of nAChR with (4-[3-(trifluor- Pore. An isoflurane dimer (colored brown in Fig. 1) occupies the omethyl)-3H-diazirin-3-yl]benzyl 1-(1-phenylethyl)-1H-imida- nAChR pore for the last 300 ns of the simulation. Each molecule zole-5-carboxylate) (TDBzl-etomidate) (16) yields labeling of in the dimer shields the other from water on one side, so that in pore-lining residues (α∶S252). Furthermore, single channel narrow regions of the water-filled pore each binding site assumes recordings of the nAChR under control conditions indicate that qualities of a hydrophobic-polar interface, like those known (5–8) openings of the channel are isolated. After exposure to isoflur- to bind volatile anesthetics. ane, the openings occur in clusters (or bursts) and are separated The two isoflurane molecules are mobile within the nAChR by brief closures. The duration of the bursts is nearly equivalent pore, with contact residues ranging from those in 6′ (a serine ring) to the open time in the control system (25–27). One interpreta- to those in 16′ (a hydrophobic ring). Mutagenesis studies (28, 29) tion of this “flickering” effect is that major conformational have indicated that mutation of α∶S100 (α∶S252) to a hydropho- changes occur on relatively similar time scales in the absence bic residue increases sensitivity to isoflurane; our results are and presence of isoflurane, but that isoflurane binds and unbinds highly consistent with that work, because we find both that the quickly to the pore, blocking ion flow and dramatically shortening 10′ position is frequently occupied by isoflurane (Fig. 2) and that the effective open time. We see binding of isoflurane to the hydrophobic contacts are preferred by isoflurane bound to the pores of both the nAChR and GLIC structures, which were pore (Fig. S3) Smaller isoflurane density peaks, associated with solved under conditions resulting in a functionally closed and the second molecule in the dimer, are observed between 6′ and open conformation, respectively. 10′ and between 13′ and 16′. Exchange between the two isoflurane molecules is frequent, despite the presence of low-probability Intersubunit. Four isoflurane molecules (colored orange in Fig. 1) regions between the discrete sites. bind to three sites in the subunit interfaces of the nAChR TM Binding to an analogous site (also by an isoflurane dimer) is domain, below the M2–M3 loop. The αδ–δ and αγ–γ interfaces observed in simulations of the GLIC channel (Fig. 1). Isoflurane are both occupied persistently by either one or two isoflurane bound to the GLIC channel (Fig. 2) also follows a strongly peaked molecules; in the ligand binding domain (LBD) these interfaces trimodal distribution with the smaller peaks in similar correspond to agonist-binding sites. In addition, one isoflurane locations as for nAChR. The most probable contact residue for molecule binds loosely to the αγ–β interface without occupying isoflurane in the GLIC pore is 13′, rather than 10′ as in the a well-defined binding site. A sample frame showing the multiply nAChR. This discrepancy is consistent with differences in pore occupied site is shown in more detail in Fig. 3. Isoflurane hydro- radius as a function of z: In nAChR, the pore is relatively wide gen bonds to nonbulk water and (in the δ and γ subunits) aspar- at 10′, whereas a similarly sized opening occurs at 13′ in GLIC agine residues located on M1 (see Fig. S3). Isoflurane is not (11). Isoflurane bound to the GLIC pore generally displays fewer observed to bind to subunit interfaces in the TMD of GLIC over hydrogen bonds with pore residues than does isoflurane bound the course of the 400 ns simulation. Reduced binding to the TMD to the nAChR pore (Fig. S7), which is likely due to the higher of GLIC is likely due to the increased packing density of GLIC hydrophobicity of the GLIC pore and the absence of serine and relative to nAChR (Fig. S1).

Brannigan et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 27, 2021 Fig. 4. Sample frame showing isoflurane bound to the center of the TMD of the αδ subunit. M1 is purple, M2 is green, M3 is blue, and M4 is cyan; cholesterol is yellow, isoflurane is colored by atom type. Hydrogen bonds are represented by yellow dashed lines.

Given that isoflurane only binds to such sites in α subunits of the nAChR, it is tempting to rule out sites in the other chains. The unexpected asymmetry of this mode of interaction across GLIC subunits suggests that the isoflurane concentration is too low to consistently bind to all five sites, but it is possible that isoflurane has a higher affinity for α subunits of the nAChR than for GLIC. (Alternatively, it is possible that fivefold symmetry of binding to Fig. 3. Sample frame showing the αγ –γ interface in the TMD. The γ subunit is red and the αγ subunit is blue, with cartoon overlay showing M2–M3 loop. GLIC would be seen if the simulation were run longer, but it Cholesterol is yellow, water is colored by atom type, and the two isoflurane seems unlikely because most isoflurane has partitioned into molecules are silver or green. the membrane by the end of the simulation.) In GABAA receptors, mutation of only one or two subunits greatly reduces anes- Intersubunit sites for isoflurane in the nAChR overlap with thetic sensitivity, suggesting that fivefold binding is not necessary what we termed the “B” hypothetical binding sites for cholesterol for clinical effects (19). Supposing exactly two isoflurane mole- in ref. 14. The present simulations demonstrate that binding of cules binding to five intrasubunit sites with equal affinity, there α cholesterol to the B site does not preclude the binding of one is only a 10% chance that both would bind to subunits. A pre- ference for α subunits appears to stem from the replacement of or more volatile anesthetics to the anesthetic site. Isoflurane rests α∶T229 with hydrophobic residues in β, δ, and γ chains; because near the cholesterol hydroxyl, although hydrogen bonding be- there is little solvation by water in these regions (Fig. S7), polar tween isoflurane and cholesterol in the B sites is minimal. As residues offer the only likely hydrogen bond acceptors. shown in Fig. 3, isoflurane can bind between cholesterol and A binding site in this region of the nAChR has not been con- the transmembrane-LBD interface, disrupting contacts between clusively pinpointed experimentally, to our knowledge. With em- – cholesterol and the M2 M3 loop. bedded cholesterol included, the site is likely too small for Significant experimental evidence is consistent with a site for occupancy by etomidate or neurosteroids, so it is not surprising anesthetics at subunit interfaces. Photolabeling of the nAChR re- that it is not labeled by TDBzl-etomidate (16). A photolabeling veals a halothane binding site at the αδ–δ interface (22) and a study of halothane binding to the nAChR (22) primarily detected TDBzl-etomidate binding site at αγ–γ (16), the occupation of labeling of aromatic residues, which are not found in the vicinity which is proposed to positively modulate the nAChR. Moreover, of the nAChR intrasubunit sites. A site for ethanol in the center residues in the GABAA and glycine receptors have been identi- of the subunit of GABAA has been considered in the absence of fied that contribute to positive modulation of those receptors, information regarding the location of various critical residues using photolabeling and mutagenesis (1, 3, 19–24); some of these (19), but the contact residues (Fig. S3) we find for isoflurane residues are thought to lie at the subunit interfaces in a site in this site are not homologous to any previously tested (using analogous to the intersubunit sites we observe, according to ex- the alignment prescribed in ref. 36). perimentally confirmed (34) homology models based on the Discussion nAChR. A similar site is occupied in simulations of halothane The simulations presented here constitute an extensive search for interacting with the nAChR (30, 35). The site is highly suggestive – anesthetic binding sites on a Cys-loop receptor; the method al- as an allosteric modulation site, as the M2 M3 loop is thought to lows for an unbiased search in which no preliminary information act in transduction of a ligand binding signal to the pore (12). regarding site location is required. Multiple binding sites for iso- α flurane on nAChR are found. Importantly, isoflurane binds as a Intrasubunit. Isoflurane binds to both subunit interiors (sites co- dimer to the pores of both nAChR and GLIC, in a configuration lored red in Fig. 1) in the intracellular half of the nAChR TMD, that would clearly obstruct ion flow. Given confirmation of this “ ” beneath cholesterol occupying C sites (14). Isoflurane is also presumably inhibitory site (which would dominate the effect of observed to bind in the same region of one GLIC subunit. All three isoflurane at concentrations for which it was occupied), as well isoflurane molecules bound to these sites share a homologous pair as the positive effect of many anesthetics on other Cys-loop re- of contact residues: α∶T229 and α∶V230 in nAChR and S212 and ceptors, the presence of a positively modulating site elsewhere in W213 in GLIC (Fig. S3). Additional contact residues are found the nAChR as suggested by ref. 16 seems increasingly likely. Our but are not consistently detected across the three instances of work is consistent with the results of many studies indicating that binding to this class of site. In particular, serine residues forming anesthetics bind to intersubunit sites in the TM domain, below the nAChR site frequently serve as hydrogen bonding partners. the M2–M3 loop. Such positive modulation could therefore occur

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1008534107 Brannigan et al. Downloaded by guest on September 27, 2021 through increased correlations between the conformation of the were taken directly from the results given in ref. 11 and used to protonate LBD and the TMD. We find additional indirect evidence that appropriate amino acids to describe the protein at pH 4.6. binding to this site positively modulates the receptor, because Both proteins were placed in 1-palmitoyl-2-oleoyl-sn-glycerol-phosphati- it is not found in GLIC, which is significantly more sensitive than dylcholine bilayers originally made using the MEMBRANE plug-in of Visual nAChR to inhibition by anesthetics (4). Molecular Dynamics (VMD) (38) and equilibrated using the procedure in Our MD simulations raise the possibility that isoflurane and ref. 39. The systems were solvated using the SOLVATE plug-in of VMD (38) and NaCl was added to a 0.15 M concentration. One thousand steps of mini- other small anesthetics partition into the cytoplasmic half of α mization were conducted and then all atoms of the protein were constrained Cys-loop receptor subunit TM domains, in addition to sites for 2 ns while the membrane healed around the protein. Subsequently, at the subunit interface. Because such sites lie in the core of isoflurane was inserted randomly into the water surrounding the protein, the domain, in the same plane that contains the hydrophobic con- with an isoflurane-to-lipid ratio of about 1∶3 for nAChR and about 1∶2 striction of the pore, and in the same subunit that confers sensi- for GLIC. Both systems comprised approximately 200,000 atoms. tivity to agonist, their occupancy may have a detectable or even physiologically significant effect on function. Such effects could Simulation Details. The simulations used the CHARMM22-CMAP force field potentially be through allosteric means, in which the occupancy with torsional cross-terms (40, 41) for proteins, CHARMM27 (42) for phospho- of the site stabilizes the closed or open conformation. Because lipids, ions, and water, the Cournia et al. model for cholesterol (43), and of the sites’ location behind M2 helices, however, it is also possible parameters developed in our group for isoflurane (33). Minimization and that occupancy directly hinders either tilting or rotation of M2 he- dynamics were conducted with the NAMD2.7b1 package (44). Periodic lices, which is likely required for pore opening. Fig. S3 lists contact boundary conditions were applied, with particle-mesh Ewald long-range electrostatics and a cutoff of 1.2 nm for Lennard–Jones potentials, with a residues that are conserved across the three observed instances of smooth switching function starting at 1.0 nm. Simulations were conducted binding, which could be targeted in mutagenesis studies. at a constant temperature of 300 K and pressure of 1 bar. Bonds involving hydrogen atoms were constrained to their equilibrium length using the Materials and Methods SHAKE/RATTLE algorithm. Multiple-timestep integration was carried out System Setup. Systems nAChR and GLIC were set up similarly. The nAChR using r-RESPA, with a base timestep of 2 fs and a secondary timestep of β –β coordinates were taken from 2BG9, missing 8 9 loops were modeled using 4 fs for long-range interactions. MODELLER (37), as in ref. 14, and cholesterol was inserted as in the full The systems were simulated for 15 ns with Cα atoms restrained with a occupancy system of that reference. Unlike the simulations reported in force constant of 1 kcal∕mol∕Å, followed by 400 ns with no restraints except ref. 14, the vestibule domain helices (MA) were removed for efficiency, 10 pseudobonds between the intracellular ends of the five nAChR M4 helices because their behavior probably cannot be modeled realistically without to mimic the missing vestibule domain.

the missing 100-residue M3–MA loop, and their removal significantly BIOPHYSICS AND decreases the amount of water required for simulation. Starting cholesterol Acknowledgments COMPUTATIONAL BIOLOGY coordinates were optimized using a self-implemented Monte Carlo algorithm that minimized van der Waals clashes while applying a global biasing This work was supported by the National Institutes of Health potential encouraging fivefold symmetry. The GLIC protein structure was through Grant GM055876 and by the National Science Founda- taken from the 3EAM Protein Data Bank entry and prepared in a similar tion through TeraGrid resources provided by the National Insti- manner as by Bocquet et al. (11). In particular, acid dissociation constants tute for Computational Sciences.

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