laroo.8iophys. raolec.Biol.,Vol. 57, pp. 59~9, 1992. Printed in Great Britain.All rightsreserved.

0079~107/92 $15.00 © 1992PergamonPresspie

CRYSTALLOGRAPHIC STUDIES OF A TRANSMEMBRANE ION CHANNEL, GRAMICIDIN A B. A. WALLACE Department of Crystallography, Birkbeck College, University of London, London WCI E 7HX, U.K.

CONTENTS I. INTRODUCTION: MEMBRANE TRANSPORT MOLECULES II. BACKGROUND: GRAMICIDIN, A POLYMORPHIC ION CHANNEL

59 59

III. CRYSTALSOFGRAM1CIDIN

61

IV. CRYSTALSTRUCTURE ANALYSESOF GRAMICID1N

64

V. ThE GRAMICIDIN PORE STRUCTURE

64

VI. ThE ION-FREE FORMS OF GRAMICIDIN

66

VII. THE GRAMICIDIN CHANNEL STRUCTURE

67

VIII. CONCLUSIONS

68

ACKNOWLEDGEMENTS REFERENCES

68 68

I. I N T R O D U C T I O N : MEMBRANE TRANSPORT MOLECULES Regulation of the flux of ions and molecules across the plasma membrane is an essential physiological process in all cells. The membrane lipid bilayer is an effective permeability barrier; it is the protein components of the membrane that provide the pathways for transport and the selectivity for specific ion types. Knowledge of the molecular structure of the polypeptides responsible for the conductance properties would greatly enhance our understanding of this process. To date, however, there is much less information available on the structures of these types of molecules than on other (i.e. soluble) proteins because of the paucity of high resolution crystallographic studies. This is largely due to the difficulty in producing crystals of the hydrophobic polypeptides which form channels. The availability of such structural information on a polypeptide for which there is also conductance, chemical modification, mutation, and spectroscopic data which bear on its functional properties, would provide an important link in our understanding of ion transport at a molecular level. Gramicidin A, the first membrane channel whose three-dimensional structure in complex with its ligands has been determined, is such a molecule. II. BAC~KGROUND: G R A M I C I D I N , A P O L Y M O R P H I C ION CHANNEL Gramicidin A is a linear polypeptide antibiotic which forms channel structures capable of transporting monovalent cations across phospholipid membranes (Hladky and Haydon, 1972). The molecule consists of 15 amino acids, with alternating L- and D- configurations, and one ethanolamine residue. It has the sequence (Sarges and Witkop, 1965); formyl-L-val-gly-L-ala-o-leu-L-ala-o-val-L-val-o-val-L-trp-D-leu-L-trpD-leu-L-trp-o-leu-L-trp-ethanolamine, Fluorescence and conductance measurements have shown that the active conducting form of the molecule in black lipid membranes and phospholipid vesicles is a dimer (Veatch et al., 1975; Veatch and Stryer, 1977). Circular dichroism, infrared, and nuclear magnetic resonance spectroscopic studies have shown that most of the forms found in solution are also 59

60

B.A. WALLACE

dimers (Veatch et al., 1974; Veatch and Blout, 1974; Arseniev et al., 1985; Bystrov and Arseniev, 1988), and crystallographic analyses have demonstrated that in a number of different crystal forms, as well, the structural unit is a dimer (Wallace and Ravikumar, 1988; Langs, 1988; Langs et al., 1991). Gramicidin A has proven to be a very polymorphic molecule, which is perhaps not too surprising, given its relatively small size. However, despite its polymorphism, it is not infinitely flexible, nor does it adopt "random" conformations. Rather, it has been found to adopt a discrete number of structural and functional forms. Circular dichroism spectroscopic studies showed that it forms several distinctly different conformations, depending on its environment (Wallace, 1983). In most organic solvents, the predominant forms are double helices (Fig. la), whereas in phospholipid vesicles the predominant forms are helical dimers

(a)

(b)

Fl~3. I. Schematic diagrams of the (a) double helix (pore) and (b) helical dimer (channel) forms of gramicidin A.

(Fig. lb), with a small fraction of the molecules in membranes being in a double helix conformation (Weinstein et al., 1979, 1980; Boni et al., 1986; Szabo and Urry, 1979; Bamberg et al., 1977; Bradley et al., 1978; Ovchinnikov and Ivanov, 1983; Bystrov and Arseniev, 1988). Because of the alternating L- and o-nature of the amino acid sequence, either right- or left-handed helices could potentially be formed. Additionally, helices with different numbers of residues/turn and stagger (alignment of the hydrogen bonding pattern between the two polypeptide chains) would be possible. Versions of all of these variants have been found for the double helix in solution, the predominant one depending on which conditions (e.g. solvent, concentration, ionic strength) are present. The double helix type folding motif has been designated the "pore" form, while the helical dimer is known as the "channel" form. The pore and channel forms are interconvertible (Wallace, 1984). In black lipid membranes, too, the molecule shows polymorphism: gramicidin produces a number of different types of conducting species, with the predominant one being that of the channel. Lower conducting states with similar mean channel lifetimes have been designated "mini-channels" (Busath and Szabo, 1988) and are believed to be of a generally similar overall fold as the channel form. Rarer conducting forms with lower single channel conductances but very long mean channel lifetimes appear to be of the pore type (Ovchinnikov and Ivanov, 1983; Durkin and Andersen, 1987; Koeppe et al., 1991). The polymorphism found in solution and in membranes is further evidenced in the solid state, as gramicidin crystallizes in a number of different crystal forms. Furthermore, since these include crystals in which the ligands, monovalent cations, are both present and absent, they provide insight into the nature of the ion-channel interactions. As gramicidin is the best characterized ion channel in terms of its conductance properties (for reviews, see Finkelstein and Andersen, 1981; Andersen, 1984; Hladky, 1987), having be~n studied extensively for more than 20 yr, it is entirely appropriate that it is now also the structurally best characterized membrane channel. This is in large part due to the

Crystallographic studies

61

crystallographic analyses that have recently emerged. These complement and extend the spectroscopic, chemical modification, and conductance data already available. III. CRYSTALS OF GRAMICIDIN Many crystalline forms of gramicidin have been reported over the years (Hotchkiss and Dubos, 1940; Hodgkin, 1949; Cowan and Hodgkin, 1953; Olesen and Szabo, 1959; Veatch, 1973; Veatch et al., 1974; Koeppe et al., 1978, 1979; Wallace, 1983; Koeppe and Schoenborn, 1984; Kimball and Wallace, 1984; Hedman et al., 1985; Wallace and Ravikumar, 1988; Langs, 1988; Wallace et al., 1990; Langs et al., 1991; Wallace and Janes, 1991). In general, these crystals grow slowly, most taking a minimum of 6 weeks to form, some taking more than 6 months. The first crystals of gramicidin (Hotchkiss and Dubos, 1940) were grown from acetone as part of the procedures initially used for isolation and purification of the molecule. The earliest crystals of gramicidin characterized by X-ray diffraction were prepared from "alcohol" solutions (Hodgkin, 1949). Subsequently, it was shown that very different crystal types are obtained from methyl and ethyl alcohols (Veatch, 1973) (Figs 2a and b; Table 1).

FlG. 2. Crystals of gramicidin formed from: (a) methanol, (b) ethanol, (c) caesium chloride and methanol--form I, (d) caesium chloride and methanol in the process of converting from form I to form II, (e) caesium chloride and methanol--form II, (f) dimyristoyl phosphatidyicholine and ethanol.

62

B. A. WALLACE TABLE 1. CRYSTALSOF GRAMICIDIN

Components Gramicidin/MeOH Gramicidin/EtOH Grami¢idin/MeOH/CsCl-I Gramicidin/MeOH/Csel-II Gramieidin/EtOH/CsCl Gramieidin/MeOH/CsSCN Gramieidin/MeOH/KSCN Gramicidin/MeOH/CsBr Gramieidin/DMPC Gramicidin/DPPC

Space group P21 P222t21 C2221 P212~2~ P2~2~2~ P212~21 P2~212~ P212t21 P222~ P222~

Unit c e l l dimensions 15.2 x 26.5 x 32.2 24.8 x 32.4 x 32.7 33.9 x 35.9 x 91.4 32.1 x 52.1 x 31.2 32.1 x 26.l × 31.2 32.3 × 53.2 x 31.8 32.1 x 51.8 x 31.0 31.8 x 51.8 x 30.7 26.2 x 27.3 x 32.7 26.8 × 27.5 x 32.8

Dimers/unit Volume/dimer cell (~3) 2 4 16 8 4 8 8 8 2 2

6464 6569 6952 6522 6535 6830 6443 6321 6720 6714

Although the crystals from these solvents were of different space groups and unit cells, the subcell dimensions and Patterson maps (Koeppe et al., 1978) were similar, which lead to the belief that the structures of the molecules in these two crystal types would be similar. However, later structural analyses, as we shall see, revealed significant differences in the structures obtained from these two solvents (Langs, 1988; Langs et al., 1991). Crystals of gramicidin have also been prepared from other alcohols, including methylpentanediol, trifluoroethanol, and n-propanol (Kimball and Wallace, unpublished results; Wallace, unpublished results). Even though gramicidin is a very hydrophobic molecule, the presence of water in the crystallization mixtures does not appear to have a major influence on the crystals obtained. Crystals from aqueous mixtures of methanol and ethanol have similar unit cells as the crystals prepared from anhydrous versions of the same solvents (Kimball and Wallace, unpublished results). Furthermore, Koeppe and Schoenborn (1984) showed that soaking crystals formed from ethanol in a 1: 1 water/ethanol mixture did not cause a change in unit cell parameters, nor a breakup of the crystals, even though neutron diffraction indicated that many of the added water molecules occupied ordered solvent positions in the crystals. In contrast, crystals produced in the presence of salts are not isomorphous with the ionfree gramicidin crystals, even though they can be prepared from the same solvent systems. In addition, soaking of ion-free crystals in mother liquor with salts added results in rapid cracking of the crystals and complete dissolution within minutes (Wallace, unpublished results), suggesting a significant conformational change must be occurring in the structure upon ion binding, as had been predicted from spectroscopic studies (Wallace, 1983). As we will see later, these predictions have been confirmed now that the structures of both the ionfree and ion-complexed forms have been determined (Wallace and Ravikumar, 1988; Langs, 1988). The ion-containing crystals themselves are rather diverse, and their nature depends on both the cation and anion type, as well as the crystallization conditions. Crystals have been formed with several different types of monovalent cations. These have been characterized in terms of unit cell dimensions (Table 1; Koeppe et al., 1979; Wallace, 1990), and the structure of one of the caesium complexes has been determined at high resolution (Fig. 3a; Wallace and Ravikumar, 1988). Potassium-containing crystals (Table 1) appear to be isomorphous with caesium chloride (form II) crystals and caesium thiocyanate complex crystals (Koeppe et al., 1979; Wallace, unpublished results), although lithiumcontaining crystals do not appear to be (Wallace, unpublished results); thallium-containing crystals are isomorphous with a different type of caesium-containing crystals (gramicidin/ caesium chloride~form I) (Wallace, 1990), and are believed, based on the unit cell dimensions, to represent a form of the molecule with a smaller helical pitch. A number of different crystal forms have also been obtained when different anions have been included in the crystallization mixtures. While caesium chloride, caesium bromide, and caesium thiocyanate crystals are apparently isomorphous (Table 1; Koeppe et al., 1978; Kimball and Wallace, 1984; Wallace, 1990; Buswell and Wallace, in preparation), single isomorphous replacement and Patterson analyses of the X-ray data (Koeppe et al., 1978;

Crystallographicstudies

63



(a)

(b)

FIG. 3. Crystal structures of (a) the gramicidin pore complex with caesium chloride and (b) the ion-free form of gramicidin (from Wallace, 1990; after Wallace and Ravikumar (1988) and Langs (1988)).

Ravikumar and Wallace, 1990) have indicated that the positions of the caesiums alon~ the length of the pores in these crystals differ considerably. It is interesting to speculate thai the different sites in the different caesium complexes might represent alternative successive binding sites the ion can occupy as it is transported through the pore. Detailed information on the natures of the different ion binding sites await the solutions of the crystal structures of the caesium bromide and caesium thiocyanate complexes. Even out of the same crystallization solution two different crystal forms of the gramicidin/caesium chloride complex are produced (Kimball and Wallace, 1984). Initially form I crystals (Fig. 3c), which are needles, come out of solution, but if left untouched, these crystals dissolve and out of the resulting two-phase system (Fig. 3d), are produced chunkier (form II) crystals (Fig. 3e), which exhibit better order, and have different unit cell dimensions (Table 1). In addition to the large effects of ions on the crystal types produced, another variable is the presence of lipid molecules. When gramicidin is incubated in the presence of dimyristoyl (DMPC) or dipalmitoyl (DPPC) phosphatidylcholine, a very different type of crystal is produced (Fig. 3f). The crystal lattice contains both lipid and gramicidin molecules (Short et al., 1987; Wallace and Janes, 1991). The solvent content of these crystals is low, and spectroscopic analyses show that the lipids in the crystals are well ordered (Short et al., 1987). It is speculated that the lipids may be occupying what would otherwise be ordered solvent molecule positions in the lattice. These crystals behave differently in the presence of salts than do the lipid-free crystals: they do not dissolve or disorder when placed in a mother liquor which has been saturated with caesium (Wallace, unpublished results). This corresponds well with the observation in solution that, unlike the pore form, the lipid-bound channel form does not change its pitch upon binding ions (Wallace et al., 1981). The diversity of morphology and unit cell parameters of the different crystals formed by gramicidin is a further manifestation of the polymorphism seen for this molecule in solution by spectroscopic and conductance methods. Interestingly, however, despite this diversity, the volume occupied by a gramicidin dimer in each of the crystal forms is nearly identical (Table 1), indicating that the solvent contents and packing densities for all the different molecular folds are similar (Wallace, 1990).

64

B.A. WALLACE

IV. CRYSTAL STRUCTURE ANALYSES OF G R A M I C I D I N Although the earliest crystals of gramicidin were first reported as long ago as 1940, this molecule has proved to be particularly recalcitrant to solution by X-ray structural analysis methods. Koeppe et al. (1978) succeeded in producing Patterson maps of several crystal forms of the polypeptide and Koeppe and Schoenborn (1984) published a low resolution (5 ,~) map showing the molecular shape based on neutron studies, but no high resolution structure showing the backbone fold had been determined until recently, when the structures of both an ion-complex and two uncomplexed forms were determined (Wallace and Ravikumar, 1988; Langs, 1988; Wallace et al., 1990; Langs et al., 1991). This is so because the molecule falls in a difficult intermediate size range for crystallographic studies: too large for the simple direct methods used for small molecules, and too small for the multiple isomorphous replacement methods traditionally used for macromolecules. The problems with the latter stem from the difficulty in producing isomorphous heavy atom derivatives of gramicidin. This is because the crystals have a very low solvent content (,-~20%), the polypeptide backbone undergoes conformational changes with almost any change in condition, and the side chain types present few sites suitable for binding of heavy atom labels. Solution of the structure of the ion-complexed form took advantage of the fact that caesium, which has a large anomalous scattering component for CuK~ radiation, can bind in the channel (Wallace et al., 1990). The anomalous dispersion and the partial structure of the caesium atoms were used to produce an initial set of phases for a 1.8 A resolution data set that was obtained from a single crystal of a gramicidin/caesium chloride complex; from this, an initial model was built (Wallace and Hendrickson, 1985; Wallace, 1986), and the structure was refined (Wallace et al., 1990; Wallace and Ravikumar, 1988). To solve the structure of the uncomplexed form from ethanol, Langs (1988) utilized a combination of direct phasing methods and molecular replacement, and both traditional tangent formula and random start phasing procedures, to produce firstly the structure of a fragment, and eventually, of the whole molecule at 0.9 A resolution. Such a combination of techniques was necessary because this molecule represents the upper limit of current day "small molecule" techniques. The form from methanol, solved at 1.5 A resolution (Langs et al., 1991), was not sufficiently similar to the ethanol structure to allow simple molecular replacement methods to be used. Rather, a trial model based, for the most part, on the polypeptide backbone found in the ethanol structure, followed by significant modification by rebuilding, produced the methanol structure, which refined only after substantial side chain disorder was included. Finally, the new crystals which contain a complex of gramicidin and phospholipids, have been the subject of stereochemical modelling studies (Wallace and Janes, 1991) in order to understand the nature of the packing and molecular structure in this crystal form. These analyses were based on the stoichiometry of the two components present, the unit cell and space group (Table 1), the molecular transform evident from the diffraction patterns, and spectroscopic studies of the crystals. The packing arrangement was derived using one half of the dimer model produced by normal mode analysis (Roux and Karplus, 1988) as the gramicidin monomer model, along with the published crystal structure of a phospholipid molecule (Hitchcock et al., 1974). This study, while not yet providing the complete structure solution, strongly suggests that the gramicidin is in its channel conformation in these crystals, and provides further evidence for the polymorphism of the molecule. V. THE G R A M I C I D I N PORE S T R U C T U R E The pore, found in the gramicidin/caesium chloride complex crystals, is a tube-like structure, approximately 26 A in length. Each tube is formed from two polypeptide chains oriented in an antiparallel manner and linked by 14 hydrogen bonds organized in a beta sheet-type pattern. The two beta strands are then wrapped into a double helix, forming an additional 14 hydrogen bonds. This results in a double helical structure, with a helix repeat of 10.4 ,~ (Fig. 3a). The pore structure is thus held together by a total of 28 hydrogen bonds, all formed from the polypeptide backbones. The hydrogen bonds are in a B 7"2 hydrogen bonding motif, but

Crystallographic studies

65

because there is also a superhelical twist present, there are actually 6.4 residues in each helical turn, rather than the 7.2 per turn suggested by the hydrogen bonding motif. A further consequence of the superhelical twist is that all of the hydrogen bonds run nearly parallel to the axis of the helix; half of them point with their dipoles in one direction and half in the opposite direction. Nearly all of them lie along the surface of the tube, the only exceptions being the ones that are involved in the ion binding sites (see below). T ~ two polypeptide chains are aligned within the beta sheet such that the amide of the sixth residue of one chain forms a hydrogen bond with the carbonyl group of the 14th residue of the other chain, and vice versa, the eighth residue forms hydrogen bonds with the 12th residue, etc. This leaves only residue 2 unpaired at the end of the sheet, as well, of course, as the odd numbered residues on each side of the sheet. When the sheet is rolled up, a second set of 14 hydrogen bonds is formed, involving the odd numbered residues (i.e. the first residue in one chain is paired with the 13th of the other chain, the 3rd with the 1lth, etc.). This means that the second set of hydrogen bonds between the two chains are offset or "staggered" by two residues, leaving the 15th residue in each chain unpaired. This stagger produces a threedimensional object with nearly flat surfaces at the ends of the pore. Because of the stereochemical nature of the amino acids in the gramicidin sequence, the double helical folding motif produces a tube with a central hole. A beta sheet formed of all L-amino acids would have side chains protruding on both sides of the sheet, and were it to be rolled up into a helix, the central pore of such a structure would be "plugged" with side chains. However, in gramicidin, which has alternating L- and D-amino acids, all side chains protrude on the same side of the sheet. So, when the sheet is rolled up, the side chains (which are all hydrophobic) are located on the exterior of the helix. The centre of the helix is not blocked by side chains. The diameter of the hole is ~ 4.9 A, hence it can easily accommodate ions. The polypeptide backbone provides a hydrophilic environment for the ions and solvent molecules inside the pore. Although many of the side chains of gramicidin are large, they pack in a way which produces a nearly uniform outside diameter (~ 16 A) along the length of the pore. The average X2 angle for the tryptophans is ~ 90 degrees, which means that the aromatic rings are not oriented perpendicular to the helix axis, but rather are lying more or less parallel to it. The tryptophans tend to interleave with average dihedral edges of -~ 75 degrees, producing a herringbone-type arrangement, as is often seen in soluble proteins. Caesium and chloride ions, as well as solvent molecules, are located in the centre of the pore, thereby providing us with an opportunity to examine pore-ligand interactions. At first glance it would seem that since the lumen of the pore and the ion binding sites are formed from the polypeptide backbone, which is relatively homogeneous throughout its length, the ions might experience this as a uniform tube in which caesiums could occupy any number of sites with similar probabilities. This is not the case, however. At equilibrium in the crystal, there are two discrete sites within the pore where caesiums bind, located, on average, 7.2 A from each end of the tube and separated by 11.6 A. While the polypeptide backbone does form a relatively uniform tube, it is somewhat distorted from its average tk, ~P angles in the regions which form the ion binding sites. The carbonyl groups nearest the caesiums are tilted towards the centre of the pore (by up to a 40 degree angle from the surface of the tube), to permit complexation. This produces a slight puckering of the chain. The cations are located in nearly symmetric positions with respect to the two polypeptide chains. For each caesium ion, the atoms which are closest are the carbonyl oxygens of the 1lth and 14th residues of one polypeptide chain (the average caesium-oxygen distance is 3.6 A). Since these residues are separated by approximately one half turn of helix, they are located on opposite sides of the pore. The closest atoms in the other polypeptide chain are the carbonyl oxygens of the second and third residues, which are located at an average distance of 3.8 A. A somewhat unexpected finding, since gramicidin is studied as a cation transporter, is that there are three chloride ions found in each pore. However, there has been some evidence for anion permeability (Eisenman et al., 1977), anion binding, and anion influence on cation permeability in the channel (Eisenman et al., 1978). Furthermore, Sung and Jordan (1988) have calculated that for the pore form of the molecule, the barrier to chloride binding is at the

66

B.A. WALLACE

entrance to the pore; if this barrier can be surmounted, chlorides should be stable in the interior of the pore. The observed presence of chlorides in the pore crystals may be more a consequence of the relatively high salt concentration used to form the crystals, however, and hence, the chlorides may be occupying sites in the crystal that would otherwise contain solvent molecules in solution. The chloride positions are considerably more variable than the caesium positions with respect to the adjacent residues in the polypeptide backbone. However, there are at least two amide nitrogens within 4/k of each of the chlorides, The distances between all caesiums and chlorides are > 5 A, which indicates they exist as individual ions. There are also a number of solvent molecules within the pore, between the ions, and especially at the ends of the pore. VI. THE ION-FREE FORMS OF GRAMICIDIN In crystals formed from ethanol in the absence of ions, gramicidin also adopts the structure of a left-handed antiparallel double helix (Langs, 1988), but this structure differs substantially in ~b, ~' angles, hydrogen bonding pattern, helical pitch, and regularity of the polypeptide backbones and tube diameter from that of the caesium complex. In this structure, there are 5.6 residues per turn, and no superhelical twist, so the hydrogen bonds lie at an angle to the helix axis (Fig. 3b). As a consequence of the smaller number of residues per turn, the tube is significantly longer (31/k) and the internal hole in this form is narrower than in the caesium complex. On average the internal diameter is 4.8 A, but it varies along the length of the tube from 3.8 to 5.5 A. The hydrogen bonding within the beta sheet (involving the even numbered residues) is similar to that in the ion complex (i.e. the sixth residue forms a hydrogen bond pair with the 14th residue, the eighth with the 12th, etc). However, the alignment of the second set of hydrogen bonds (the odd numbered residues), formed in the rolled up sheet, is different, due to the difference in pitch of the helix. That means that in this form, the first residue of each chain forms a pair of hydrogen bonds with the 15th residue of the other chain, the third with the 13th, etc. Hence, the stagger of the chains is zero in the ion-free form and thus different than in the caesium complex. In this case, none of the odd numbered residues is unpaired; in three dimensions, however, this sort of alignment results in the ends of the pore being more uneven, or "ragged". The polypeptide backbone of the ion-free form has substantial irregularities or "bulges" along its length, resulting in a large variation in the ~b, W angles found along the chain. As a result of this and the larger pitch of this helix (on average 10.9/k) relative to that found in the caesium complex, the central hole is too narrow in some regions to accommodate ions. Indeed, in this crystal form, neither ions nor solvent are found in the centre of the molecule, although a number of ordered solvent molecules are found elsewhere in the crystal. Modelling studies (Langs, 1988) have suggested that in some of the regions of the tube, where the bulges occur, there is sufficient room for a potassium ion to bind; however, that apparently does not happen, since potassium-containing crystals are isomorphous with caesium-containing crystals rather than ion-free crystals. Like the caesium-complex form, in the ion-free form from ethanol, the side chains are all located on the periphery of the molecule and the tryptophans tend to lie roughly parallel to the helix axis. In contrast, in the ion-free form from methanol, the tryptophan side-chains also lie at the periphery, but in this case they are oriented nearly perpendicular to the helix axis (X2,-~0 degrees) (Langs et al., 1991). This is the major difference between the methanol and ethanol structures and has been attributed to differences in packing arrangements, which permit considerable pi stacking interactions between dimers in the lattice. There are also some differences in the backbone structures of the two alcohol forms, which result in the methanol form being much more uniform along its length. Its average pore diameter is 5.1 A, with the narrowest part being 4.3 A in diameter, and the largest bulge being 5.5 A. However, the helical pitches, handedness, hydrogen bonding patterns, and lengths of the two ion-free forms are virtually identical. It is clear that the presence of ions has a major impact on the folding of the polypeptide chains. Although the ion-complexed and ion-free forms adopt the same sort of folding motif,

Crystallographic studies

67

the left-handed double helix, and are each held together by 28 hydrogen bonds, the differences in the relative alignments of their polypeptide chains mean that the process of refolding upon ion binding must involve the breakage and reformation of many hydrogen bonds. One model for this (Salemme, 1988) suggests a relative unwinding, in a corkscrewtype motion, that although topologically simple, would require breakage of all 28 hydrogen bonds. Another possible mechanism would involve breakage and reformation of only the 14 hydrogen bonds involved in creating the rolled up structure, and not those within the sheet, but would require a complete uncoiling and then recoiling of the sheet to form a tube. Both mechanisms would present a large energy barrier to the process, although the net energy differences between the initial and final forms should be small since the same number of hydrogen bonds would ultimately be both broken and reformed during the process. VII. T H E G R A M I C I D I N C H A N N E L S T R U C T U R E Recently, crystals have been prepared of a lipid complex of gramicidin (Table 1) in which the structure of the molecule is clearly different from that in any of the crystal forms prepared in the absence of lipids (Wallace and Janes, 1991). These crystals contain two lipid molecules and one gramicidin monomer in their asymmetric unit. The monomers are related in the crystal lattice by symmetry operators which produce the dimeric unit. The cell dimensions and space group of the crystals are consistent with an end-to-end association of gramicidin monomers and a bilayer-like motif of lipid molecules, with extensive regions of lipid-lipid contact between the gramicidin dimers (Fig. 4a). The partial interdigltation of lipid chains

-

? ;N (a)

(b)

F1G. 4. Proposed packing arrangement of gramicidin and lipid molecules in the grarnicidin/dipalmi-

toyl phosphatidylcholine(DPPC) crystals (fromWallaceand Janes, 1991). (Fig. 4b) required by the packing motif, explains why the dimensions of crystals made with fatty acid chains of different length (DMPC and DPPC) do not vary very much. A priori, it had been thought that the cell dimension corresponding to the normal to the lipid "bilayer" plane should increase by ~ 5 A, with the increase from 14 (DMPC) to 16 (DPPC) carbon chain fatty acids. However, the observed difference of < 1 A, is consistent with an increased overlap of the chains. Interestingly, the crystals from the longer chain lipids are better ordered, perhaps due to the more extensive lipid-lipid interactions. Although the structure of the gramicidin-lipid co-crystal has not yet been solved, the X-ray data available thus far can be satisfactorily modelled with a channel-like helical dimer structure containing 6.3 residues per turn.

68

B.A. WALLACE VIII. CONCLUSIONS

I n s u m m a r y , the i n f o r m a t i o n thus far d e r i v e d from the v a r i o u s crystal structures of g r a m i c i d i n gives us the first d e t a i l e d view o f a n ion channel in c o m p l e x with its ligands. Such high r e s o l u t i o n c r y s t a l l o g r a p h i c s t r u c t u r a l i n f o r m a t i o n has p r o v e n to be c o m p l e m e n t a r y to the wealth o f d a t a a v a i l a b l e from spectroscopic, c o n d u c t a n c e , a n d chemical m o d i f i c a t i o n studies o n this molecule. C o m b i n e d , these m e t h o d s give the m o s t extensive insight yet into the s t r u c t u r e / f u n c t i o n r e l a t i o n s h i p s involved in the process of i o n t r a n s p o r t across m e m b r a n e s . I n the future, studies o n a n u m b e r of o t h e r i o n a n d lipid c o m p l e x e s of g r a m i c i d i n s h o u l d serve to further enhanc~ o u r u n d e r s t a n d i n g of h o w the molecule changes its structure in response to the b i n d i n g a n d t r a n s p o r t of ions a n d in the presence of lipid molecules. ACKNOWLEDGEMENTS I t h a n k the students, p o s t d o c s , a n d colleagues with w h o m I have w o r k e d on these studies: N. O. Buswell, J. D. C a l l a h a n , A. K. D u n k e r , M . F a u s e l , W . A. H e n d r i c k s o n , R. W . JaDes, M . R. K i m b a l l , K . R a v i k u m a r a n d G . A. W o o l l e y . This w o r k has been s u p p o r t e d over the years b y the Biophysics P r o g r a m of the U n i t e d States N a t i o n a l Science F o u n d a t i o n (currently g r a n t DMB88-16981). REFERENCES ANDERSEN,O. A. (1984) .4. Rev. Physiol. 46, 531-548. ARsm~mv,A. S., BARSUKOV,I. L., BYSTROV,V. F., LOMIZE,A. L. and OvcmNNmov, Y. A. (1985) FEBS Lett. 186, 168-174. BAMaERG,E., APELL,H. J. and ALPES,H. (1977) Proc. narD..4cad. Sci. U.S.,4.74, 2402-2406. BONI,L. T., CONNOLLY,A. J. and KLEINFELD,A. M. (1986) Biophys. J. 49, 122-123. BRADLEY,R. J., URRY,D. W., OKAMOTO,K. and RAPAKA,R. (1978) Science 200, 435436. BUSATH,D. and SzABo,G. (1988) Biophys. J. 53, 689~595. BVSTROV,V. F. and ARSENIEV,A. S. (1988) Tetrahedron 44, 925-940. COWAN,P. M. and HOI~GKIN,D. C. (1953) Proc. R. Soc. Set. B 141, 89-92. DURKIN,J. T. and ANt~ERS~.N,O. S. (1987) Biophys. J. 51,451a. Ei~ENMAN, G., SANDBLOM,J. and NEHER, E. (1977) In Metal-Ligand Interactions in Organic Chemistry and Biochemistry (eds. B. PULLMANand N. GOLDBLUM),pp. 1-36. EISENMAN,G., SANDI~LOM,J. and NEHER,E. (1978) Biophys. J. 22, 307-340. FINKELSTEIN,A. aad Ar~ERSEN,O. S. (1981) J. Membr. Biol. 59, 155-171. H ~ M ~ , B., HODG~ON,K. O., HELLIWELL,J. R. and PAPIZ,M. Z. (1985) PNAS 82, 7604-7607. H~TCHCOCK,P. B., MASON,R., THOMAS,K. M. and SHIPLEY,G. G. (1974) PNAS 71, 3036-3040. HLADKY,S. B. (1987) In Ion Transport Through Membranes (eds. K. YAGIand B. PULLMAN),pp. 213--232. HLADKY,S. B. and HAYDON,D. A. (1972) Biochim. biophys. Acta 274, 294-312. HOIX~KIN,D. C. (1949) Cold Sprino Hath. Syrup. quant. Biol. 14, 65-78. HOTCHKISS,R. D. and Duaos, R. J. (1940) J. biol. Chem. 132, 791-792. KII~mALL,M. R. and WALLACE,B. A. (1984) `4nn. N.Y. Acad. Sci. 435, 551-554. KOEPPE, R. E. and SCHOENBORN,B. P. (1984) Biophys. J. 45, 503-508. KOEPPE, R. E., HOImSON,K. O. and STRYER,L. (1978) J. molec. Biol. 121, 41-54. KOEPPE, R. E., BERG,J. M., HODGSON,K. O. and STRYER,L. (1979) Nature 279, 723-725. KOEPPE, R. E., II, GREATHOUSE,D. V., PROVIDENCE,L. L. and ANDERSEN,O. S. (1991) Biophys. J. 59, 319a. LANES,D. A. (1988) Science 241, 188-191. LANG$,D. A., SMITH,G. D., COURSEILLE,C., PRECIGOUX,G. and HOSPITAL,i . (1991) PN`4S ~ , 5345-5349. OLESEN,P. E. and SZABO,L. (1959) Nature 183, 749-750. OVCHINNIKOV,YU. A. and IVANOV,V. T. (1983) In Conformation in Biology (eds. R. SRINIVASANand R. H. SARMA), pp. 155-174. RAVIKUMAR,K. and WALLACE,B. A. (1990) Biophys. J. 57, 488a. Roux, B. and KARPLU$,i . (1988) Biophys. J. 53, 297-309. SALEMME,F. R. (1988) Science 241, 145. SARGES,R. and WITKOP,B. (1965) J. `4m. Chem. Soc. 87, 2011 2020. SHORT,K. W., WALLACE,B. A., MYEI~,R. A., FODOR,S. P. A. and DUNKER,A. K. (1987) Biochemistry 26~ 557-562. SUNG, S.-S. and JORDAN,P. C. 0988) Biophys. J. 54, 519-526. SZABO,G. and URRY,n. W. (1979) Science 203, 55-57. VEATCH,W. R. (1973) Ph.D. Thesis, Harvard University, Boston, MA. VEATCH,W. R. and BLOUT,E. R. (1974) Biochemistry 13, 5257-5264. VEATCH,W. R. and STRYER,L. (1977) d. molec. Biol. 113, 89-102. VEATCH,W. R., FOSSEL,E. T. and BLOUT,E. R. (1974) Biochemistry 13, 5249-5256. VEATC-'~,W. R., MATHmS,R., EISENJ~tw,~M. and STRYER,L. (1975) J. molec. Biol. 99, 75-92. WALLACE,B. A. (1983) Biopolymer~a2~2~t 139~7t-~,02. WALLACE,B. A. (1984) Biophys. J. 45, 114-116. WALLACE,B. A. (1986) Biophys. J. 49, 295-306. WALLACE,B. A. (1990) .4. Rev. Biophys. biophys. Chem. 19, 127-157. WALLACE,B. A. and HENDRICKSON,W. A. (1985) Biophys. J. 47, 173a.

Crystallographic studies

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Crystallographic studies of a transmembrane ion channel, gramicidin A.

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