USA. Vol. 70, No. 6,pp. 1836-1840, June 1973. Conformations of the Li-Antamanide Complex and Na-[Phe4, Val6JAntamanide. Complex in the Crystalline State.
Proc. Nat. Acad. Sci. USA Vol. 70, No. 6, pp. 1836-1840, June 1973
Conformations of the Li-Antamanide Complex and Na-[Phe4, Val6JAntamanide Complex in the Crystalline State (Roentgen-ray analysis/cyclic peptides/cis- and trans-peptide bonds/ penta-coordinate sodium and lithium complexes/ion transport)
ISABELLA L. KARLE*, JEROME KARLE*, Th. WIELANDt, WOLFGANG BURGERMEISTERt, HEINZ FAULSTICHt, AND BERNHARD WITKOPt * Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20390; tMax-Planck-Institut Fur Medizinische Forschung, Heidelberg, Germany; and t National Institutes of Health, Bethesda, Maryland 20014
Contributed by Bernhard Witkop, April 10, 1973
ABSTRACT Antamanide, a cyclic decapeptide isolated from the poisonous mushroom Amanita phalloides, preferably complexes with Na+, but in less polar solvents, e.g., acetonitrile, also with Li+ or K+. The selectivity of complexation makes it an important model for the study of conformational requirements of ion binding. The conformations of the lithium antamanide complex and the Na-[Phe4, Val6ljantamanide complex have been established by x-ray diffraction analyses of single crystals. The two compounds are isostructural, but not isomorphous. The complexes are folded into a globular shape "with an approximate 2-fold axis. Two of the peptide linkages are in the cis conformation, Pro2-Pro3 and Pro7-PrqP. There are only two intramolecular hydrogen bonds. Four C=O groups have their 0 atoms directed inward to form four Li-0 or Na-0 ligands. The fifth ligand to the metal ion is provided by a solvent molecule. The conformation found in the crystalline state is different from any of the conformations proposed for the sodium antamaiide complex in solution on the basis of nuclear magnetic resonance data.
The conformation of cyclic polypeptides has been the subject of many studies and considerable speculation. The first instructive example illustrative of the conformational complexity of cyclic peptides was afforded by the crystal structure analysis of cyclic hexaglycyl (1), which demonstrated the existence of four different, well-ordered conformers side-byside in each unit cell. One of the conformers had two intramolecular hydrogen bonds (subsequently designated by other authors as 1 4 hydrogen bonds), while the other three conformers participated only in intermolecular hydrogen bonding. Another example of the unpredictability of the conformations of cyclic peptides is that of c-Gly-Gly-DAla-D-Ala-Gly-Gly. In the crystalline state, the compound exists as a single conformer analogous to the c-6 Gly conformer with two internal hydrogen bonds (2), while a nuclear magnetic resonance (NMR) analysis together with minimum energy calculations (3) indicate that in solution c4Gly-2D-Ala is a flexible molecule rapidly interconverting between conformations, none of which approximates the crystalline structure.
Abbreviation: NMR, nuclear magnetic resonance. 1836
Antamanide is a cyclic decapeptide, isolated from the highly 8 9 10 1 2 Pro-Phe-Phe-Val-Pro (all -) Pro-Phe-Phe-Ala-Pro
7
6
6
4
3
poisonous mushroom Amanita phalloides (4). This cyclopeptide is particularly fascinating in that it forms complexes with Na+, Ca+ + Li , and K+ ions. Selectivity and stability of the complexes change with solvent (5), the most stable complexes being formed with Na+ and Ca++ ions. Hence the conformation may be of considerable significance for the biological activity of antamanide, the antidote to the liver toxin phalloidin, although it does not act itself as an ion carrier through cell membranes (6). Different models have been proposed for free and complexed antamanide on the basis of extensive nuclear magnetic resonance, circular dichroism, optical rotatory dispersion measurements (7-9), and minimum conformational energy calculations (10). On the basis of these techniques, the molecule in solution has been interpreted to possess a 2-fold axis of rotation; the sodium complex to have a rigid conformation independent of the nature of the solvent, and the. uncomplexed molecule to exist as two or more conformers that rapidly interconvert depending upon the composition of the medium. The model of Ivanov et al. (7) for the sodium complex contains exclusively trans peptide bonds, an octahedral array of carbonyl oxygen atoms (from residues 1, 3, 5, 6, 8, and 10) coordinating to the sodium ion and four intramolecular hydrogen bonds. The most favorable model for sodium antamanide proposed from a recent redetermination by Patel (9) on the basis of proton and '3C NMR studies and confirmed by Tonelli (10) through minimum conformational energy calculations, contains two cis-peptide bonds, one at Val' adjacent to Pro2 and the other at Phe6 adjacent to Pro7. The cavity is lined with eight carbonyl oxygen atoms, four of which (from residues 4, 5, 9, and 10) coordinate with the alkali ion. An x-ray diffraction analysis of single crystals of the lithium-antamanide and of the Na-[Phe4, Val6jantamanide complexes show that the conformations of the alkali complexes differ from any of the models considered previously.
Proc. Nat. Acad. Sci. USA 70
(1978)
Li-Antamanide and Na-[Pl4e4, Val6]Antamanide Complexes
1837
FIG. 1. Stereodiagram of the Li-antamanide complex drawn from the experimentally determined coordinates. The Li+ ion is black and the backbone of the cyclic decapeptide is bold face. Dotted lines indicate hydrogen bonds and solid lines show the Li-O ligands. A molecule of CH3CN, which forms the fifth ligand to the Li+, is omitted for clarity.
EXPERIMENTAL Crystals of the lithium complex were grown by dissolving 11.474 mg of antamanide in 1 ml of acetonitrile (0.01 M) and by mixing this with a solution of 0.869 mg of LiBr dissolved in 1 ml of acetonitrile (0.01 M). The solution was heated to 800 (with a Dewar flask serving as a water bath) and gradually cooled to room temperature. Crystallization occurred overnight. It is essential to keep the crystals suspended in their mother liquor, otherwise they deteriorate rapidly. For the purpose of collecting x-ray diffraction data, a crystal was sealed in a thin-walled, boron glass capillary containing a drop of the mother liquor, which created a sufficient vapor pressure of acetonitrile to prevent the disintegration of the crystal during the collection of diffraction data, usually over a period of 9 days. The crystal used for data collection was a clear stout prism with good faces. The cross-section was 0.23 mm X 0.36 mm, and the length was 0.7 mm. The space group is P21, with a = 11.912 i 0.004 A, b = 23.206 i 0.006 A, c = 13.884 i 0.003 A, and f3 = 1100 45' + 04'. A total of 5945 independent reflections were recorded with copper radiation; however, only those reflections with IFol > 5, 4960 in all, were used for the least-squares refinement. A four-circle automatic diffractometer was used in the 0-20 scan mode to- collect the diffracted intensities. The coordinates of the Br- ion were readily determined from a Patterson map computed with (IEhklI2 - 1) as coefficients where the Ehk1 values are the normalized structure factors (11). The remaining 94 atoms in the lithium-antamanide complex plus three solvent molecules (CH2CN) were located in three successive E-maps. Phases for the E-maps were obtained by use of the phases for JEl > 1.5, if jEcall > klEobsI, from the known fragment of
the structure, and extension of the phase determination by means of the tangent formula (12, 13) to all IEl > 1.1. Coordinates and anisotropic thermal factors were refined by means of least-squares for the Br- ion and the six oxygen atoms in residues 2, 4, 5, 7, 9, and 10, since a large amount of anisotropy was evident in the difference map for these atoms. For the remaining 89 atoms, isotropic thermal factors were used. At present, the R-factor is 10.3%. Although refinement is not completed, and perhaps all the solvent molecules have not yet been found, the conformation of the lithium-antamanide complex is well documented. Bond lengths and bond angles are near the expected values for peptide groups and side chains. CONFORMATION OF THE LITHIUMANTAMANIDE COMPLEX A three-dimensional view of the complexed molecule is represented by the stereodiagrams in Figs. 1 and 2. The con-
formational features include approximate 2-fold rotation symmetry with the 2-fold axis passing through the alkali ion. Thus, residues 2 and 7, 3 and 8, and 5 and 10 are closely related by molecular symmetry; however, the symmetry cannot be exact, since the side chains on 1 and 6 and on 5 and 9 are quite different. This approximate 2-fold symmetry has also been apparent in the NMR spectra (7, 9, 10). One unusual feature is the occurrence of two cis-peptide bonds. These are associated with each pair of prolyl groups and appear between Pro2-Pro3 and Pro7-Pro8. Patel (9) has suggested that two cis and two trans X-Pro peptide bonds are present in antamanide. The most likely model presented by Patel, consistent with the interpretation of the NMR spectra and low-energy cyclic peptide conformations, contains cis-
FIG. 2. Stereodiagram of the Li-antamanide complex seen at right angles to the view in Fig. 1. The CH3CN solvent molecule has been included, but the two phenyl groups from Phe5 and Phe10 have been omitted for clarity.
1838
Ch.mistry: Karle et al.
Proc. Nat. Acad. Sci. USA 70 (1978)
bonds are formed involving the following residues:
.O
oes
Ala4
Phe5
Phe9
Phelo
Pro3
Phe6
Pro8
Val'
90
-if 0
-90
FIG. 3. A plot of the * and t conformational angles for Liantamanide. The numbers enclosed in circles denote the ten peptide groups. Roman numerals refer to peptide groups in c-4Gly-2D-Ala (2).
Val'-Pro2
and
Phe6-Pro7, a result that is at
variance with the structure determined in the crystalline state. Patel (9) also considered a model with cis bonds at Pro2-Pro3 and Pro-7Pro8 which, however, was different in most other respects from the structure that has now been established in the crystal. Moreover, in the final analysis he preferred a 1,6-cis structure for the Na+ complex. The remainder of the peptide segments are planar and trans. The backbone of the decapeptide ring is folded into a shape resembling that of a periphery of a saddle with the alkali ion located near the mathematical saddle point. The four phenyl groups of the phehylalanine residues are folded toward the backbone so as to make the molecule more globular and compact. The proton on N(6) is directed toward 0(3) with an 0(3) NH(6) distance of 3.05 and similarly the proton on N(1) is directed toward 0(8) with an 0(8)... NH(1) distance of 3.00 A. Thus two intramolecular hydrogen
The hydrogen-bond lengths are larger than those usually found for intermolecular O... HN bonds (2.70-2.95 A) and not as large as the two intramolecular 1 -- 4 hydrogen bonds in c-4Gly-2D-Ala (3.04 and 3.16 A) (2). Conformational angles are shown in Table 1 and plotted in the 1 versus 40 map in Fig. 3. The points are clustered in two distinct regions. The region near I = 0 corresponds to residues Ala4, Phe5 and Phe9, Phe'0, the residues involved in the 1 4 intramolecular hydrogen bonds. This same region near v = 0 also contains the v and I values for the residues Gly', GlyvI in c-4Gly-2D-Ala that form a 1 -- 4 intramolecular hydrogen bond (2). In other words, a 1 -- 4 intramolecular hydrogen bond can have the same conformational angles whether the component residues are Gly,Gly as in c-4Gly21-Ala, or AlaPhe and Phe,Phe, as in the complexed antamanide. These experimental results differ from the predictions from calculations for the lowest-energy conformation (14) where it had been concluded that (12 -60°, I2 -40°, 4'3 '~ - 1200, and I3 --. 606 (new convention) for the 1 4 intramolecular hydrogen bond involving amino-acid residues other than glycine. Four carbonyl oxygen atoms, 0(1), 0(3), 0(6), and 0(8), are directed toward the interior of the molecule and coordinate with the lithium ion with an average Li ... 0 distance of 2.11 A. The four oxygen atoms are nearly coplanar with the lithium ion 0.4 A above the oxygen plane, as illustrated in Figs. i and 2. The antarnanide complex is cup-shaped in this region, and the opening is plugged by one molecule of the CH3CN solvent with the nitrogen atom directed toward the lithium to make a fifth ligand with a Li ... N distance of 2.07 A. Thus the lithium ion has five ligands to atoms arranged in a square pyramid. In inorganic materials, the coordination scheme for lithium is either tetrahedral with Li ... 0 distances of -'1.98 A or octahedral with Li ... 0 distances of --.2.16 A (15). In the crystal of lithium succinate, the lithium is surrounded by four oxygen atoms arranged in a slightly deformed tetrahedron with Li... 0 distances of 1.95 A (16). Other
FiG. 4. Stereodiagram of the Na-[Phe4, Val6]antamanide complex. A molecule of C2H5OH, which forms the fifth ligand to the Na+, is omitted for clarity. All the stereodiagrams were drawn by computer with the help of a program prepared by C. K. Johnson, Oak Ridge
National Laboratory.
Proc. Nat. Acad. Sci. USA 70
(1978)
Li-Antamanide and Na-[Phe4, Val6]Antamanide Complexes
1839
TABLE 1. Conformational angles* for the lithium-antamanide complex j
1
2
3
4
5
6
7
8
9
10
-1ji Oj
-115
-83 147
-67
-78 - 15
176
144 -3
144
-173
-123 139 -171
-69
178
-84 -6 -178
-74
- 14
wi
-65 139 -3
-176
172
-88 7 173
138
* The convention followed is that proposed by the IUPAC-1UB Commission on Biochemical Nomenclature (1970) Biochemistry 9, 3471. In the fully extended chain 4'j = 'Ij = wj = 1800. The older convention differs from this one by i 1800.
alkali complexes, such as those of the cyclic depsipeptides valinomycin or enniatin B, or the Na+ complex of ATP (17) are all hexa-coordinate (18). The cage complex of lithium cryptate is hexa-coordinate (19), while the polyether alkali crown complexes are hexa-, octa- (sodium), and even decacoordinate (potassium) (20). Hence the penta-coordinate lithium of the antamanide complex is quite novel, and the Li ... 0 distances are intermediate between those for the tetrahedral and octahedral coordinations. For larger metal complexes five ligands are unexceptional, e.g., iron pentacarbonyl, the unoxygenated Fe++ hemo- and myoglobins, or the thallium salt of the antibiotic grisorixin, while zinc in carboxypeptidase is bound to three amino acids and one water molecule in a somewhat distorted tetrahedral complex (18). The lithium-antamanide complex is a very compact molecule. The rigidity of the decapeptide ring is enhanced by the formation of six smaller rings, i.e., two 10-membered rings formed by the two internal hydrogen bonds, two 10-membered rings that include the pairs of prolyl residues formed by ligands to the lithium ion, and two 7-membered rings (including the H atom in the internal hydrogen bond) formed by ligands to the lithium ion that encompass residues 1 and 6, respectively. Atoms 0(3) and 0(8) play multiple roles in that they form ligands to the lithium ion as well as participate in intramolecular hydrogen bonding. The exterior of the upper half of the molecule (as seen in Fig. 2) is predominantly hydrophobic. The surface is composed of hydrogen clusters from the four pyrrolidine rings, the isopropyl moiety from Val', and the phenyl moiety from Phe6. In addition, the channel through which the lithium ion is admitted to the interior of the molecule is closed by one CH3CN solvent molecule with its CH3 group completing the hydrophobic surface. The lower half of the molecule has three phenyl groups (the phenvl groups in Phe5 and Phe'0 have been omitted in Fig. 2) folded upward. As a consequence, the polar inner region along with several NH groups are shielded from the solvent, while four carbonyl oxygen atoms, 0(4), 0(5), 0(9), and 0(10), as well as two NH groups, N(4) and N(9), protrude from the surface and presumably act as hydrophilic sites. CONFORMATION OF THE SODIUMANTAMANIDE COMPLEX A crystal structure analysis was also performed on the sodium complex of the [Phe4, Val6] analog of antamanide, which still 8 9 10 1 2 Pro-Phe-Phe-Val-Pro
Pro-Val-Phe-Phe-Pro 7 6 5 4 3
has
a
(all L-)
relatively high antitoxic activity at
a
dose of 1.5-2
mg/kg as compared to 0.5 mg/kg for antamanide (4, 21). The sodium complex crystallizes in the orthorhombic space group P212121, whereas the lithium complex belongs to the monoclinic space group P21. Furthermore, the solvent used for the lithium complex is acetonitrile, while ethanol was used for the sodium complex. In spite of these differences, the two complexes are isostructural, as can be seen by comparing Figs. 1 and 4, except for the different side chains at residues 4 and 6. The fifth ligand to the sodium ion is provided by the 0 atom from the solvated ethyl alcohol; hence the 5-fold coordination to the sodium ion closely resembles that of the lithium ion. Since the conformation of the sodium complex in solution, according to the spectral data, is independent of solvent and since the lithium-antamanide and the Na-[Phe4, Val6]antamanide complexes are essentially isostructural in the crystalline state, the conclusion is justified that the conformation of the metal complex in solution should not differ significantly from that in the crystalline state. The crystal structure analysis of uncomplexed antamanide is now in progress. Uncomplexed antamanide is not isomorphous with either the lithium or sodium complexes. Spectroscopic data (4, 9, 10) suggest that the uncomplexed molecule exists in at least two conformations in solution. Hence the possibility of disorder in the crystal must be anticipated. 1. Karle, I. L. & Karle, J. (1963) Acta Crystallogr. 16, 969-975. 2. Karle, I. L., Gibson, J. W. & Karle, J. (1970) J. Amer. Chem. Soc. 92, 3755-3760. 3. Tonelli, A. E. & Brewster, A. I. (1972) J. Amer. Chem. Soc. 94, 2851-2854. 4. See Wieland, T. (1972) "Properties of antamanide and some of its analogues," in Chemistry and Biology of Peptides, ed. Meienhofer, J. (Ann Arbor Science Publishers, Ann Arbor, Mich.), p. 377 and references therein. 5. Faulstich, H. & Wieland, T. (1972) Peptides, Proc. XI European Peptide Symposium, Bad Reinhardsbrunn, DDR. 6. Ovehinnikov, Yu. A., Ivanov, V. T., Barsukov, L. I., Melnik, E. I., Oreshnikova, N. A., Bogolyubova, N. D., Ryabova, I. D., Miroshnikov, A. I. & Rimskaya, V. A. (1972)
Experientia 28, 399-401.
7. Ivanov, V. T., Miroshnikov, A. I., Abdullaev, N. D., Senyavina, L. B., Arkhipova, S. F., Uvarova, N. N., Khalilulina, K. Kh., Bystrov, V. F. & Ovchinnikov, Yu. A. (1971) Biochem. Biophys. Res. Commun. 42, 654-663; Ovchinnikov, Yu. A., Ivanov, V. T., Bystrov, V. F. & Miroshnikov, A. I. (1972) "Conformation of antamanide in non-polar solvents," in Chemistry and Biology of Peptides, ed. Meienhofer, J. (Ann Arbor Science Publishers, Ann Arbor, Mich.), p. 111. 8. Faulstich, H., Burgermeister, W. & Wieland, T. (1972) Biochem. Biophys. Res. Commun. 47, 975-983. 9. Patel, D. (1973) Biochemistry 12, 667-688. 10. Tonelli, A. E. (1973) Biochemistry 12, 689-692. 11. Hauptman, H. & Karle, J. (1953) "Solution of the phase problem. I. The centrosymmetric crystal," in A.C.A.
1840
12. 13. 14. 15. 16.
Chemistry: Karle et at.
Monograph No. 3 (Polycrystal Book Service, Pittsburgh), p. 34. Karle, J. & Hauptman, H. (1956) Acta Crystallogr. 9, 635651. Karle, J. (1968) Acta Crystallogr. Sect. B 24, 182-186. Venkatachalam, C. M. (1968) Biopolymers 6, 1425-1436. International Tables for X-Ray Crystallography (1962) (The Kynoch Press, Birmingham, England), Vol. III, p. 258. Klapper, H. & Kuppers, H. (1973) Acta Crystallogr. Sect. B 29, 21-26. Compare: Wudl, F. & Smith G. M. (1972) 164th ACS National Meeting, New York, August 28-Sept. 1, Abstract ORGN 138 and Wudl, F., personal communication.
Proc. Nat. Acad. Sci. USA 70 (1973) 17. Kennard, O., Isaacs, N. W., Motherwell, W. D. S. & Watson, D. G. (1972) in Symposium on Purines, Theory and Experiments, eds. Pullman, B. & Bergmann, E. D., p. 114. 18. Compare (1973) Inorganic Biochemistry, ed. Eichhorn, G. (Elsevier Publishing Co., Amsterdam-London-New York), Vol. I. 19. Moras, D. & Weiss, R. (1973) Acta Crystallogr. Sect. B 29, 400-403. 20. Bush, M. A. & Truter, M. R. (1971) J. Chem. Soc. B, 745-750. 21. Wieland, Th., Dungen, A. v. & Birr, Ch. (1971) FEBSLett. 14, 299-300.
