Protein Science (1992), I , 1073-1077. Cambridge University Press. Printed in the USA. Copyright 0 1992 The Protein Society

Crystallization of proton channel peptides

BRETT LOVEJOY,’ KARIN S. AKERFELDT,’ WILLIAM F. DEGRADO,’ AND DAVID EISENBERG’ I Molecular Biology Institute, University of California, Los Angeles, California 90024-1570

Biotechnology Department, DuPont Merck Pharmaceutical Company, P.O. Box 80328, Wilmington, Delaware 19880-0328

(RECEIVED February 24, 1992; REVISED MANUSCRIPT RECEIVED March 27, 1992)

Abstract

Crystals have been grown of two similar peptides that form ion-conducting channels in diphytanoyl phosphati- dylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2-trifluoroetha- nol. Crystals of one of the peptides have been characterized by X-ray diffraction, and X-ray data have been measured to 2.3 A resolution. Earlier it was proposed that the ion-conducting channels formed by these peptides consist of four peptides associated as a parallel a-helical tetramer. On the basis of the space group and unit cell dimensions of the crystals, a packing scheme for the peptide is proposed that is consistent with a tetrameric channel.

Keywords: crystallization; ion channels; peptides

The passage of ions through membrane-bound protein channels is fundamental to many physiological processes such as the transmission of electrical signals through the nervous system (Hille, 1984). Before we can fully under- stand the function of this important class of proteins, their structures must be determined. Analysis of the se- quences of a number of cloned channel proteins has shed light on their architecture. Channel proteins contain mul- tiple domains, each of which appears to contain several membrane-spanning amphiphilic a-helices. In structural models of these membrane-spanning a-helical domains, polar side chains protrude from one face of the helix, and the polar faces of several parallel a-helices form a hydro- philic ion-conducting pore (Greenblatt et al., 1985; Guy & Seetharamulu, 1986).

To gain insight into the mechanisms by which a-helices can aggregate and conduct ions, two simple amphiphilic peptides - H2N-(LSLLLSL)3-CONH2 and H,N-(LSL- Aib-LSL)3-CONH2 - were designed to form tetrameric coiled-coil structures in which the serines of each mem- ~. -

Reprint requests to: David Eisenberg, Molecular Biology Institute, University of California, Los Angeles, California 90024-1570.

Abbreviations: 4-MBHA, 4-methylbenzhydrylamine resin; AcOH, acetic acid; ADA, N-(2-acetamido)-2-iminodiacetic acid); Aib, a-ami- noisobutyryl; Boc-LEU-PAM, Boc-leucine-4-(oxymethyl)-phenylacet- amidomethyl resin; CD, circular dichroism; DIC, I ,3-diisopropylcar- bodiimide; DMF, N, N’-dimethylformamide; DIEA, N, N’-diisopro- pylethylamine; DMSO, dimethylsulfoxide; HPLC, high performance liquid chromatography; HOBT, hydroxybenzotriazole; MeOH, meth- anal; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanoI.

ber of the tetramer face each other, forming a hydrophilic channel (Lear et al., 1988; DeGrado & Lear, 1990). CD spectroscopy of the two peptides solubilized in phospho- lipid vesicles showed that they were highly a-helical, and conductance measurements demonstrated that they formed proton channels in diphytanoyl phosphatidylcholine bi- layers. Larger cations such as Li+, Na+, K+, or Cs+ were impermeable, suggesting that the ion conduction pore has a diameter of no more than 1 A. Consistent with the pep- tide design, molecular modeling studies of the peptide in trimeric, tetrameric, pentameric, and hexameric helical bundles indicated that both a trimer and a tetramer have a channel diameter of less than 1 A, whereas the penta- meric and hexameric models have channel diameters of 5 and 8 A. In these models, side chains of neighboring he- lices pack together in a “knobs-into-holes” fashion typi- cal of coiled coils (Crick, 1953; Karle et al., 1990a; O’Shea et al., 1991).

The conductance measurements and modeling studies (Lear et al., 1988; DeGrado & Lear, 1990) suggest that the peptide forms either a trimer or tetramer in phospho- lipid membranes. Here we describe a packing scheme for one of the peptides that is consistent with the tetramer but not the trimer. The present study is of peptide crystals grown from a TFEIwater mixture. The difference in the apolar environment of the conductance studies and the polar environment of the present crystallographic study needs to be considered when assessing the relevance of our model to the conducting channels.

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1074 B. Lovejoy et al.

Results

Crystallization

An H2N-(LSLLLSL)3-CONH2 mother liquor consisting of 1 mg/mL peptide was made in TFE and an H2N- (LSL-Aib-LSL)3-CONH2 mother liquor consisting of 2 mg/mL peptide was made in a 5050 (v/v) TFE:water solution. Crystals were grown using a modified form of vapor diffusion in which TFE-solubilized peptide in a drop was equilibrated against a precipitating solution in a well. For H2N-(LSLLLSL)3-CONH2 crystals, the well solution consisted of 30 mM NH4S04, 36% (w/v) TFE, and 50 mM ADA titrated to pH 6.5 with NaOH. For H2N-(LSL-Aib-LSL)3-CONH2 crystals, the well solution consisted of 40 mM KCl, 42% (w/v) TFE, and 40 mM ADA titrated to pH 6.5 with NaOH. The low salt con- centration present in these well solutions served only to improve crystal quality and was not necessary for crys- tallization. As the concentration of water increased in the peptide drop, hexagonal crystals measuring approxi- mately 0.10 x 0.10 x 0.02 mm formed. Some crystals of H2N-(LSL-AiB-LSL)3-CONH2 grew as large as 0.15 x 0.15 x 0.04 mm (Fig. 1).

X-ray characterization

Crystals of H2N-(LSL-Aib-LSL)3-CONH2 diffracted X- rays significantly better than those of H2N-(LSLLLSL)3- C0NH2, and as a consequence, we were able to determine the space group and unit cell dimensions of H2N-(LSL- Aib-LSL)3-CONH2 but not of H2N-(LSLLLSL)3-CONH2.

Fig. 1. A crystal of H2N-(LSL-Aib-LSL)3-CONHz measuring 0.15 x 0.15 x 0.04 mm.

A 16" screened precession photograph of the hkO zone of an H2N-(LSL-Aib-LSL)3-CONH2 crystal distinctly re- vealed sixfold symmetry. X-rays diffracted to the edge of the photograph, corresponding to a resolution of 2.8 A. A 12" screened precession photograph of the hOf zone showed inversion symmetry but not mm symmetry, indi- cating that the crystal system is trigonal. In addition, the reflection condition 001: I = 3n was observed, indicative of a 31 or 32 axis. A 12" screened precession of the hhf zone revealed inversion symmetry, confirming that the space group for this crystal is P3121 or its enantiomer P3,21. Unit cell dimensions are a = b = 40.93 A and c = 51.89 A. Calculation of the Mathews number suggests that two molecules in the asymmetric unit are likely with (V, = 2.91 A3 Da" [Mathews, 19681). One molecule in the asymmetric unit would give V, = 5.82 A3 Da", a value that is unusually large. Three molecules in the asymmet- ric unit would give V, = 1.94 A3 Da" and would require unusually tight packing.

X-ray data collection

A total of 9,601 reflections with Z/a(Z) 2 1 .O were mea- sured from a crystal of H2N-(LSL-Aib-LSL)3-CONH2 using an R-axis IIC imaging plate (Rigaku USA, Dan- vers, Massachusetts). Symmetry-related reflections were averaged to give 2,179 unique reflections constituting 87% of the unique data to 2.3 A resolution. The final Rmerge for all data with Z/a(Z) L 1 was 0.087, where R,, = E; I( Zi ) - Zi I /Ej Zi and (Z;) is the average of Ii over all symmetry equivalents.

Discussion

The larger size and better diffraction of H2N-(LSL-Aib- LSL)3-CONH2 compared to H2N-(LSLLLSL)3-CONH2 crystals suggest that Aib increases the rigidity of H2N- (LSL-Aib-LSL)3-CONH2. Karle and Balaram (1990) have also found that the incorporation of Aib into peptides improves their crystallinity. Assuming that H2N-(LSL- Aib-LSL)3-CONH2 is an a-helix, as CD experiments in- dicate, it can be approximated as a cylinder roughly 3 1 A long and 8-9 A in diameter. Because there are probably two peptides in the asymmetric unit, 12 cylinders must pack into a 40 x 40 x 51-A cell while obeying P3,21 or P3221 symmetry. To address the question of whether these restrictions allow for the packing of a tetramer as designed, various packing models were studied.

In the first model (Model I, Fig. 2) the two peptides of one asymmetric unit, A and B, have their helical axes par- allel to the trigonal axis. Three pairs of helices are related by the 31 axis. A twofold axis that is perpendicular to the 31 axis generates an additional set of paired helices that are antiparallel to the first set. In this packing ar- rangement neighboring dimers join to form an a-helical tetramer. However, this tetramer is inconsistent with the

Proton channel crystallization 1075

h

Fig. 2. Two models for the packing of H2N-(LSL-Aib-LSL),-C0NH2 into the space group P3,21. Model I: The two peptides of the asymmet- ric unit, A and B, are parallel to the 3, axis. Shading is used to empha- size that the twofold axis requires the peptides on one half of the unit cell to be antiparallel to the peptides on the other half of the unit cell. In this model, peptide A can be either parallel or antiparallel with re- spect to peptide B. This packing arrangement does not allow a parallel a-helical tetramer. Model 11: Peptides A and B are parallel to one an- other, with one lying directly above the other as viewed down the three- fold screw axis. Shading is used to emphasize that the twofold axis no longer interferes with the formation of a parallel or-helical tetramer. The filled circles are used to designate the amino termini of the helices. Sym- metry notation is from Henry and Lonsdale (1965).

original design of parallel helices because the twofold axis forces the two helix pairs to run antiparallel. This pack- ing arrangement might be unfavorable because there is little stabilizing lattice contact between neighboring a - helical tetramers. In the second model (Model 11, Fig. 2) peptides A and B are parallel to one another, with one ly- ing directly above the other as viewed down the threefold screw axis. In this model the twofold rotation axis gen- erates a second dimer, which together with the original di- mer forms a parallel tetramer. This parallel tetramer is consistent with the original design of a tetramer with ro- tational symmetry. The extent to which the members of this tetramer interact is uncertain since the peptides can overlap the 31 axes and consequently move away from twofold axis symmetry mates. The two models represent two extremes of allowed packing. In fact, any number of packings ranging from the first model, with the dimer parallel to the threefold screw, to the second model, with the dimer perpendicular to the threefold rotation axis, are allowed. Both models are based on a tetramer and do not allow for the formation of a trimer.

Thus, the crystal space group is consistent with a struc- ture that would be similar to the designed tetramer. How- ever, it should be appreciated that the peptide was designed to form tetramers in membranes in the presence of an applied transmembrane potential - conditions that are very different from the crystallization conditions. A transmembrane electrical potential should stabilize a par- allel tetramer if the N-terminal, positively charged ends of its a-helical dipoles face the negative side of the bi- layer. On the other hand, in the absence of a voltage an antiparallel tetramer would also be probable. Further, peptides in a transmembrane orientation are constrained to interact as helical bundles. Such constraints would not be present in the absence of a membrane. Finally, the peptides were designed to place their polar Ser side chains on the interior of the structure when placed among the apolar fatty acid acyl chains of the membrane. In a po- lar solvent such as TFE/water it is possible that the po- lar ser residues will be directed toward the exterior of the structure rather than be buried as modeled for the pro- ton channel. Thus the crystal structure of the peptide may not represent the channel-forming state. Nevertheless, it should provide insights into the mechanisms by which a- helices are stabilized and interact to form higher-order structures.

To determine the crystal structure we must determine the phases of the diffracted X-rays. Direct methods, the usual approach for smaller molecules, may not be appli- cable to our crystal form. The reasons are that our data are limited in resolution to 2.3 A and the number of non- hydrogen atoms in the asymmetric unit, 302, exceeds the current threshold of most approaches to direct methods. Isomorphous replacement will be attempted, and if the resolution of data can be extended, so will molecular re- placement.

Materials and methods

Sitting drop vapor diffusion

Concave glass cylinders 14 mm in height and 10 mm in di- ameter (Perpetual System Corporation, Rockville, Mary- land) were washed in soap in water with a toothbrush and then soaked for 20 min in a 2.5% (v/v) solution of Aqua- Si1 (Pierce, Rockford, Illinois). The rods were then rinsed, dried, and sealed into each well of a Linbro tray (Flow Laboratories, McLean, Virginia) by applying a small drop of chloroform to the bottom of the wells and then dropping a rod into each well. A thin film of vac- uum grease (Dow Corning, Midland, Michigan) was ap- plied to the rim of each well. After allowing the seal to dry for 1 h, a total of 1 mL of precipitant was pipetted into each well of the Linbro tray. H2N-(LSLLLSL)3-CONH2 drops were formed by mixing 5 pL of mother liquor with 5 pL of well solution in the concave groove at the top of the glass rod. Similarly, H2N-(LSL-Aib-LSL)3-CONH2 drops were formed by adding 15 pL of well solution to

1076 B. Lovejoy et al.

8 pL of mother liquor and 10 pL of water. The wells were sealed immediately after the mother liquor had been added by applying a 22 x 22 x 2-mm glass coverslip (Fisher Scientific, San Francisco, California) to the rim of vacuum grease. All mother liquor solutions were cen- trifuged for 10 min at 14,000 rpm immediately before use. All trays were incubated at 30 "C for 2 weeks before crystals were harvested.

Synthesis of H2N-(Leu-Ser-Leu-Aib-Leu-Ser-Leu)3- CONH2

The (LSL-Aib-LSL)3 peptide was synthesized by a step- wise solid-phase protocol employing standard Boc-chem- istry (DeGrado & Lear, 1990). The problem with the synthesis, however, is that it provides a product that con- tains trailing impurities in the HPLC profile that are exceedingly difficult to remove. Earlier attempts to as- semble the peptide by solid-phase segment condensation of protected heptamers failed due to difficulties encountered in the synthesis of Fmoc-Leu-Ser(Bz1)-Leu-Aib-Leu- Ser(Bz1)-Leu-(0-t-Bu) on the oxime support (DeGrado & Lear, 1990). Instead, we found that the Aib-containing peptide is readily obtained by solution segment conden- sation of unprotected heptamers. The procedure involves the preparation of two separate heptamers, Boc-LSL- Aib-LSL-COOH and H2N-LSL-Aib-LSL-CONH2, and yields a highly pure product. The procedure has the added advantage that it is amenable to scale-up.

H2N-(Leu-Ser-Leu-Aib-Leu-Ser-Leu)-CONH, ( I ) The peptide was synthesized using 4-MBHA resin, ac-

cording to the synthetic protocol described by DeGrado and Lear (1990). After completion of the synthesis, the resin (2.2 g) was treated with HF/m-cresol 1O:l (22 mL) for 1 h at 0 "C. After removal of H F under reduced pres- sure, the product mixture was washed with diethyl ether (3 x 25 mL), and the peptide extracted from the resin with 10% AcOH (2 x 20 mL), glacial AcOH (2 x 20 mL), and 10% AcOH (2 x 20 mL). The filtrate was lyophilized to a powder (0.65 g), which was redissolved in MeOH. The isolated product was collected as a beige solid after precipitation with diethyl ether (0.636 g, 75% yield); MS(FAB) m/z (expected): 729.49 (MH+); MS(FAB) m/z (found): 729.60 (MH+).

H2N-(Ser-Leu-Aib-Leu-Ser-Leu)-COOH (2) The peptide was synthesized on Boc-Leu-PAM resin

and isolated as described for compound 1. The product, obtained in 75% yield as a white fluffy powder after ly- ophilization, was directly taken on to the next reaction: MS(FAB) m/z (expected): 617.39 (MH+); MS(FAB) m/z (found): 617.55 (MH+).

Boc-(Leu-Ser-Leu-Aib-Leu-Ser-Leu)-COOH (3) DIEA (0.70 mL, 4.04 mmol) and Boc-Leu-OSu (0.66 g,

2.02 mmol) were added to a solution of hexamer 2 (1.23 g,

1.68 mmol). The reaction mixture was allowed to stir at 22 "C for 12 h before it was concentrated in vacuo and pu- rified by HPLC on a C18 reversed-phase column, eluting with a linear gradient of 45-75Vo B (flow rate: 10 mL/min; injection size: 100 mg) to furnish 888 mg (64% isolated yield) of a white solid: MS(FAB) m/z (expected): 830.52 (MH+), 852.45 (M + Na+); MS(FAB) m/z (found): 830.68 (MH+), 852.66 (M + Na+).

H2N-(Leu-Ser-Leu-Aib-Leu-Ser-Leu)2-CONHz (4) HOBT (41 mg, 0.301 mmol) and DIC (47 pL, 0.301

mmol) were added to a stirred solution of Boc-protected peptide 3 (100 mg, 0.121 mmol) in DMF (270 pL). After 10 min of reaction time the white slurry, containing pre- cipitated urea, was cooled to 0 "C and amine 1 (85 mg, 0.100 mmol) in DMF:MeOH 3:l (280 pL) and DIEA (52 pL, 0.301 mmol) was added in one portion. The ice bath was removed and the reaction mixture was warmed to 22 "C. After 15 h of reaction time at pH 4-5, addi- tional HOBT (28 rng, 0.2 mmol) and DIC (30 pL, 0.2 mmol) were added to the reaction mixture. After 1 h the crude product was applied to a column (2.5 x 75 cm) packed in LH-20 resin, and the product was eluted with MeOH. Fractions 38-60 (4 mL each) were pooled and concentrated in vacuo to furnish 131 mg (79% yield) of product. An aliquot was subjected to analytical HPLC on a PRP-1 column, eluting with a linear gradient of 20- 100% B over 80 min. The major peak was observed at a retention time of 58 min. The Boc-protected 14-mer pep- tide (50 mg, 30.2 pmol) was subjected to 25% TFA in CH2Cl2 (1.0 mL). After 30 min of stirring at 22 "C the reaction mixture was concentrated under reduced pres- sure, and residual TFA was removed with repeated evap- orations with DMF. The product (retention time: 38 min, under the same analytical HPLC conditions as described above) was obtained in quantitative yield as a clear film and was directly taken on to the next reaction.

H2N-(Leu-Ser-Leu-Aib-Leu-Ser-Leu)3-CONH2 (5) HOBT (12 mg, 91 pmol) and DIC (14 pL, 91 pmol)

were added to a stirred solution of the Boc-protected pep- tide 3 (30 mg, 36 pmol) in DMSO (100 pL). After 5-10 min of reaction time the slurry was cooled to just above its freezing temperature, the amine 4 (530 rnmol) dissolved in DMSO (200 pL) was added, and the pH was adjusted to 4-5 with DIEA (this required altogether approximately 100 pL). After 1 h of reaction time at 22 "C, additional HOBT (21 mg, 156 pmol) and DIC (20 pL, 128 pmol) were added. After 15 h of reaction time the product mix- ture was concentrated in vacuo to a solid that was treated with 100% TFA (1 .O mL) for 15 min at 22 "C. The reac- tion mixture was worked up and purified by chromatog- raphy on an LH-60 resin, as described for the preparation of peptide 4. The product-containing fractions were pooled and concentrated to a white solid. The peptide was purified on a preparative PRP-1 reversed-phase col- umn under isocratic conditions (88% B) to provide 13 mg

Proton channel crystallization 1077

(20% isolated yield for the segment condensation reaction sequence): MS(FAB) m / z (expected): 2,175.5 (M + Na+); MS(FAB) m / z (found): 2,175.3 (M + Na+).

General

TFE was purchased from Eastman Kodak. ADA, NaOH, KCl, and NH4S04 were purchased from Sigma. Aquasil was purchased from Pierce Chemical. Protected amino acids were obtained from Bachem, Inc., Fluka, or Ad- vanced ChemTech. The 4-MBHA resin (substitution level: 0.7 meq/g resin) and Boc-Leu-PAM resin (substi- tution level: 0.65 meq/g resin) were purchased from Bachem, Inc., and Advanced ChemTech, respectively. LH-20 and LH-60 Sephadex were purchased from Phar- macia. HPLC was carried out on CI8 preparative (250 x 22 mm; Vydac), and PRP-1 analytical (5 p ; 50 x 4.5 mm; Hamilton) or preparative (250 x 21.5 mm), columns. Sol- vents A and B were 0.1 Yo TFA in H 2 0 and 0.1 Yo TFA in CH3CN:H20 9: 1, respectively.

Acknowledgments

We thank Isabella Karle for suggesting the incorporation of Aib to improve the crystallinity of the Aib peptide and Dan Ander- son for his help with photography. We also thank Thanh C. Le

for her assistance in the crystallization of H2N-(LSL-Aib- LSL),-CONH2, and Rob Houghton for technical assistance.

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