J. Mol. Biol. (1991) 217, 625-627

Co-crystals of Gramicidin

A and Phospholipid

A System for Studying the Structure of a Transmembrane Channel B. A. Wallace’T*t and Robert W. Janes’ 1 Department of Chemistry and Center for Biophysics Rensselaer Polytechnic Institute Troy, NY 12180, U.S.A. * Department of Crystallography Birkbeck College University of London, London, U.K. (Received 27 September 1990; accepted 26 October 1990)

Single crystals of a complex of gramicidin A, a transmembrane channel-forming polypeptide, and dipalmitoyl phosphatidylcholine, a phospholipid, have been prepared and characterized by X-ray diffraction. They belong to space group P222,, with unit cell dimensions a= 26.8 A, 6 = 27.5 A, c = 32.8 A. The asymmetric unit appears to be a complex of one gramidicin monomer and two phospholipid molecules. The unit cell dimensions, space group, and chemical composition are compatible with lipids packing in a bilayer-like motif, and an end-to-end association of gramicidin monomers to form a functional dimeric unit. The crystals diffract to 2 A and are suitable for structural studies by single-crystal X-ray analysis. This represents the first example of a well-ordered crystalline channel complexed with lipids, and solution of its structure may give insight into mechanisms of ion transport across membranes.

“channel”, is an N-terminal-to-N-terminal helical dimer (Urry, 1971). Gramicidin has been crystallized in a variety of forms in the absence of lipids (Hodgkin, 1949; Veatch, 1973; Koeppe et al., 1978; Kimball & Wallace, 1984; Wallace, 1990), and the crystal structures of two of these forms, a gramicidin/ cesium complex that adopts the “pore” conformation (Wallace & Ravikumar, 1988; Wallace et al., 1990) and an uncomplexed form (Langs, 1988), have been determined. Preliminary studies suggested that crystalline complexes of gramicidin and the lipid dimyristoyl phosphatidylcholine could be formed (Kimball & Wallace, 1981), but all were poorly ordered. By variation of the gramicidin/lipid ratio, lipid type, solvent, precipitant and temperature, conditions have now been found that produce well-ordered crystals of a gramicidin/phospholipid complex. These crystals should permit us to examine the structure of the “channel” conformation, which the molecule adopts in the presence of lipid molecules, and which is the dominant conducting form of the molecule. Gramicidin (ICN Nutritional Biochemicals), a

Gramicidin A is a linear polypeptide antibiotic that forms monovalent cation-specific channels in membranes (Hladky & Haydon, 1972). It has the sequence (Sarges & Witkop, 1965): Formyl-n-Val-Gly-r-Ala-n-Leu-n-Ala-n-Val-rVal-n-Val-L-Trp-n-Leu-L-Trp-n-Leu-n-Trp-n-LeuL-Trp-NHCH,CH,OH. Fluorescence and conductance measurements have demonstrated that the molecules associate to form dimers (Veatch & Stryer, 1977). Circular dichroism spectroscopy (Wallace, 1983) has shown that gramicidin adopts distinctly different conformations in membranes and in organic solvents, and that the conformation in membranes is relatively independent of lipid-to-protein ratio (Wallace, 1986). The predominant conformation of gramicidin in organic solvents is the double helix, which has been designated the “pore” structure. Spectroscopic and conductance measurements suggest that the major in phospholipid membranes, the component t Author addressed.

to whom

all correspondence

should

be

625 0022-2836/91/040625~3

$03.00/O

0 1991 Academic Press Limited

3. A. Wallace and B. W. Janes

626 mixture

of 80% gramicidin

A, 6 y0 gramicidin

ethanol and heated to 55°C for ten minutes. To some samples, deionized water was added to make a 2% solution (v/v) and the specimens left to crystallize at 20°C in the dark by very slow evaporation over a period of at least six months, with minimal disturbance. The gramicidin to lipid ratio in the resulting crystals was determined by washing individual crystals in cold solvent, followed by dissolution of the crystals in methanol. The grami-

B

and 14% gramicidin C (variants with Phe and Tyr, respectively, at position 11) was recrystallized from

Dipalmitoyl phosphatidylcholine was ethanol. purchased from Calbiochem-Boehring and used without further purification. Crystals were prepared by batch crystallization as follows: 50.0 mg of gramicidin A and 50.0 mg of dipalmitoyl phosphatidylcholine were dissolved in 1 ml of absolute

(b)

Figure 1. Diagrams showing a possible packing motif for the gramieidin and Lipid molecules in this crystal form. (a) View down the helix axis (in the a*c* plane); and (b) view perpendicular to the helix axis (in the a*b* plane).

Communications cidin content was determined by measuring the optical density at 282 nm, using an extinction coefficient of 22,000; the lipid content was determined by phosphate assay (Fiske & Subbarow, 1925). The molar ratio of gramicidin-to-lipid in the washed crystals was 1 : 2, which is somewhat different from the input ratio, and suggests the formation of a specific complex. Precession photographs of these crystals show a diffraction pattern consistent with space group P222,. Unit cell dimensions are a = 26.8 8, b=27.5 A and c=32.8 A (1 A=@1 nm). Assuming one gramicidin monomer and two phospholipids per asymmetric unit, gives a V, value of 1.79 A3/Da, which is near the low end of the range of those commonly found for soluble proteins, but similar to those found for small proteins (Matthews, 1968). Other gramicidin crystals also pack very tightly and have low solvent contents. In this case, the lipid may be occupying some of what would otherwise be “solvent” volume. The unit cell dimensions and space group of this co-crystal form are different from those of crystals prepared from ethanol in the absence of lipid (Koeppe et al., 1978; Wallace, 1986); the vibrational spectra of these crystal forms are also very different (Short et al., 1987), suggesting that the gramicidin conformation in the two crystal forms may not be the same. The unit cell dimensions, space group and chemical composition of the co-crystals are compatible with lipid molecules packing in a bilayer-like motif and gramicidin monomers in an end-to-end dimer, if the two gramicidin monomers are related by one of the 2-fold axes. One such possible arrangement of lipids and gramicidins consistent with the data is shown in Figure 1. This packing arrangement was derived using as the gramicidin monomer model conformation model one-half of the “channel” produced by normal mode analysis (Roux & Karplus, 1988) and the published crystal structure of dilauryl phosphatidyl ethanolamine (Hitchcock et al., 1974). With reference to Figure 1(a) in this model, neighboring lipids in the horizontal direction have opposing orientations, while those in the vertical direction have the same orientation, thus resulting in head-to-tail packing. It has been established that this packing arrangement does not result in any steric overlaps, but there is a partial interdigitation of the fatty acid chains (see Fig. l(b)). These co-crystals are good candidates for a highresolution structure determination for several reasons: they diffract strongly, although the crystals tend to be rather small (maximum dimension approx . -0.1 mm), they are very stable in the X-ray beam, there are reflections measurable by Edited

627

diffractometry to approximately 2.0 A, and Raman spectroscopy suggests that the lipid fatty acid chains have a very regular structure (no gauche bonds) (Short et al., 1987), so the lipids may be well ordered in the crystals. Knowledge of this structure should give us insight into the transmembrane transport activity of this well-characterized ion channel and provide the first view of a lipid-polypeptide channel complex. We thank Benoit Roux for providing us with a copy of the coordinates of the model for the gramicidin channel. This work was supported by NSF grant DMB8816981. A portion of this work was done when one of the authors (B.A.W.) was the recipient of a Senior International Fellowship (TWO 1563) from the Fogarty International Center of the National Institutes of Health. Coordinates of the model complex are available from the authors upon request.

References Fiske, C. & Subbarow, 375400. Hitchcock, P. B., Mason, G. G. (1974). Proc. 3036-3040. Hladky, S. B. 8: Haydon,

Y. (1925). J. Biol. Chem. 66, R., Thomas, K. M. & Shipley, Nat. Acad. Sci., U.S.A. 71, D. A. (1972). Biochim. Riophys.

Acta, 274, 294-312. Hodgkin,

D. C. (1949). Cold Spring Harbor Symp. Quant.

Biol. 14, 65-75. Kimball,

M. R. & Wallace, B. A. (1981). Acta. Crystallogr.

sect. A, 37, ~50. Kimball, M. R. & Wallace, B. A. (1984). Ann. N. l’. Acad. sci. 435, 551-554. Koeppe, R. E., Hodgson, K. 0. 8: Stryer, L. (1978). J. Mol. Biol. 121, 41-54. Langs, D. A. (1988). Science, 241, 1888191. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Roux, B. & Karplus, M. (1988). Biophys. J. 53, 297-309. Sarges, R. & Witkop, B. (1965). J. Amer. Chem. Sot. 87, 201 l-2020. Short, K. W., Wallace, B. A., Myers, R. A., Fodor, S. P. A. & Dunker, A. K. (1987). Biochemistry, 26, 557-562. Urry, D. W. (1971). Proc. Nat. Acad. Sci., U.S.A. 68. 672-676. Veatch, W. R. (1973). Ph.D Thesis, Harvard University, Boston, MA. Veatch, W. R. & Stryer, L. (1977). J. Mol. Biol. 113, 89-102. Wallace, B. A. (1983). Biopolymers, 22, 397-402. Wa~llace, B. A. (1986). Biophys. J. 49, 295-306. Wallace, B. A. (1990). Annu. Rev. Biophys. Biophys. Chem. 19, 127-157. Wallace, B. A. & Ravikumar, K. (1988). Science, 241, 182-187. Wallace, B. A., Hendrickson, W. A. & Ravikumar, K.

(1990). Acta Crystallogr. sect. B, 46, 440-446.

by R. Huber

Co-crystals of gramicidin A and phospholipid. A system for studying the structure of a transmembrane channel.

Single crystals of a complex of gramicidin A, a transmembrane channel-forming polypeptide, and dipalmitoyl phosphatidylcholine, a phospholipid, have b...
3MB Sizes 0 Downloads 0 Views