GOPINATH K-ARTHA, KOTTAYIL I. VARUGHESE, AND SABURO AIMoTo. Center for Crystallographic .... and the perchlorate. w. 4520. Chemistry: Kartha et aL ...
Proc. .Nat Acad. Sci. USA Vol. 79, pp. 4519-4522, July 1982 Chemistry
Conformationof 'eyCo(-L-Pro-Gly-)3 and its Ca2' and
Mg2 complexes
(cyclic hexapeptide/ionophore/octahedral coordination/x-ray crystallography)
GOPINATH K-ARTHA, KOTTAYIL I. VARUGHESE, AND SABURO AIMoTo Center for Crystallographic Research, Roswell Park Memorial Institute, Buffalo, New York 14263
Communicated by Elan R - Blout, Apri 12, 1982
ABSTRACT The synthetic hexapeptide cyclo(-L-Pro-Gly-)3 is an ionophore that shows interesting conformational changes upon binding metal ions. X-ray crystallographic studies of this peptide show that when it is crystallized from an ethanol/ethyl acetate mixture the ring takes up an asymmetric conformation containing one cis peptide bond. In crystals of a Ca"s complex, the cation is sandwiched between two peptide molecules that differ markedly in conformation. However, both exhibit threefold symmetric forms, with all six peptide bonds in.the molecule occurring in the usual trans conformation. The Ca"' is octahedrally surrounded by six glycyl carbonyl oxygens from the two peptides at an average distance of 2.26 A and can easily be released by the disruption -of the peptide sandwich. In the magnesium complex, the peptide forms a 1:1 complex with the ion. The Mg2 is octahedrally coordinated to three glycyl carbonyls and three water oxygens. The average coordination distance between magnesium and the peptide oxygens is 2.03 A and that between magnesium and water oxygen-is 2.11 A. The two peptide molecules in the asymmetric unit have similar conformations and have approximate threefold symmetry.
Table 1. Crystal data
Space group Cell constants, a, b, and c in A, g in0
P3212 P63 a = 11.379(3) a = 12.366(1) c = 32.93(1) c = 20.830(1)
Z No. of peptide molecules in the asymmetric unit 20,m. Cu Ka, °
6
4
P21
a = 12.677(2) b = 12.340(5) c = 21.502(2) P = 92.69(1) 4
1
2/3
120
150
2 150
1,633
6,107 10
No. of reflections used in structure determination and 1,478 refinement Final R factor, % 8
Naturally occurring cyclic peptides such as enniatin (1-3) and valinomycin (4, 5) are known to bind ions and mediate their transport across natural and prepared biological membranes; these peptides have been the subject of extensive investigations. The conformational changes that facilitate ion binding and transport can be studied in considerable detail by using x-ray crystallographic techniques, and such studies yield insight into the mechanism of ion binding and transport through membranes at the molecular level. These studies and. the interpretation of-the results are much simplified by the use of simple model peptides that mimic these properties and can easily be synthesized in the laboratory. One such synthetic peptide that mimics the ion binding properties of antamanide (6) and enniatin is. the hexapeptide cyclo(-L-Pro-Gly-)3 [hereinafter denoted (PC)3], which has been extensively studied by Blout and co-workers by spectroscopic and computed potential energy techniques (7, 8). From their studies they concluded that (PG)3 conformation varies significantly writh the nature of the medium. In nonpolar medium as well as when forming complexes with cations, (PG)3 takes up a threefold symmetric conformation, and in polar solvents this changes to an asymmetric conformation with one of the peptide bonds in the cis configuration. Even though crystallographic results of a few cyclic hexapeptides have been reported, most of these contain two f-turns, with the peptide rings having at least an approximate twofold or inversion symmetry. The threefold nature of the chemical sequence of (PG)3 and the existence of alternating proline residues exert conformational restrictions that make the usual dou-
C21H30N606. C21H30N60,6, %/Ca(C104)2. i/X(C104)2. %/2H20 3H20
C2jH30N606.
3/H20
(PG)35Mg
(PG,)3'Ca
(PG)3 Molecular formula
8
ble 3-turn structure with intramolecular hydrogen bonds impossible for this hexapeptide. We report here the conformational features of this peptide and the stoichiometry and geometry of two of its cation complexes.
EXPERIMENTAL (PG)0 was synthesized according to the procedure described by Deber and Blout (9) and crystals were obtained from ethanol/ ethyl acetate mixture by slow evaporation. The crystals of calcium and magnesium complexes with the peptide were obtained from aqueous medium by the addition of calcium or magnesium perchlorate to the solution. The diffraction data were measured at room temperature on an Enraf Nonius automatic diffractometer. The crystal data are given in Table 1. The crystal structure of both (PG£)3 and (PG)3-Ca were solved by multisolution techniques (10). (PG)3'Mg had 97 nonhydrogen atoms in an asymmetric unit and all attempts to solve the structure by direct methods were unsuccessful. The cation positions were located from the sharpened Patterson function and the structure was solved by a number of cycles of structure factor and Fourier calculations. The positional and thermal parameters were refined by block diagonal least-squares techniques. The crystallographic details and atomic parameters will be published elsewhere. (PG)3 Molecule. Some of the main features of (PG)3 are shown in Fig. 1. This is not a symmetric structure and has no /3-turns
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Abbreviation: (PG)3, CyClO(-L-PrO-Gly-)3. 451.9
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Chemistry: Kartha et aL
Proc. NatL Acad. Sci. USA 79 (1982)
FIG. 1. Stereo view of (PG)3 molecule crystallized from a polar medium. The a-carbons are numbered 1-6. The structure is asymmetric with one Gly-Pro peptide bond (4-5) in the cis configuration. The carbonyls of the first and fourth residues are linked by a hydrogen-bonded water bridge involving three water molecules (W1-3). The water molecule W2 sits on a twofold axis.
or internal hydrogen bonds (11, 12) characteristic ofcyclic hexapeptides. Five of the peptide bonds in the ring occur in the usual trans conformation, and one ofthe Gly-Pro bonds is cis. Though cyclic di- and tripeptides can be fbimed only with all peptide links cia (13, 14), in larger peptides the ring closure can be achieved with all trans peptide units. Even though cia peptides have been noted to occur in many other cyclic peptides (15-19), they are very rarely observed in the crystallographic structures of cyclic hexapeptides. For (PG)3 such an asymmetric structure has indeed been suggested earlier by Blout and colleagues (7). Another interesting feature of the crystal structure is the formation of a dimer about the crystallographic twofold axis by strong N-H--O hydrogen bonds between N2 and 02 with a nitrogen--oxygen distance of 2. 77 A, This asymmetric conformer is stabilized by hydrogen bonds involving three water molecules linking carbonyls 01 and 04 (Fig. 1). The main chain as well as the proline ring torsion angles of this structure are given in Table 2. Peptide Ca Complex. With calcium, (PG)3 forms a 1:2 complex such that the Ca"+ is sandwiched between two peptide molecules (Figs. 2 and 3). The peptide sandwich sits on the threefold axis of the unit cell, -and thus .the crystal structure indeed uses the threefold symmetry of the molecular complex. One of the perchlorate anions is also located on this threefold axis. The calcium ion is octahedrally coordinated by glycyl carbonyls belonging to the two peptides, at an average distance of 2.26 A. The individual conformations ofthe two peptides forming the sandwich (call them A and B) show considerable differences from each other (Table 3). Their conformations are also different from the (PG)3 conformation of the uncomplexed molecule described earlier as well as from the symmetric 3--y-turm structure proposed (7) for uncomplexed (PG)3 in nonpolar solTable 2. Torsion angles (in degrees) of the (PO)3 molecule Residue / w no. Residue XO Xl X2 X3 X4 I 5 11 -23 25 --19 Pro -56 153 .179 2 91 -125 -174 Gly 3 -78 -8 -170 -15 34 -41 31 -10 Pro 4 5 6
Gly
2 90 .159 0 -77 -10 170 -8 13 -15 -10 Gly -96 -163 -178 4), 4, and w are the main-chain torsion angles (20). Xo, Xi X2, X3, and X4 are the intraring torsion angles of the proline ring. They denote the angles about N-CA, C]-CA', Ce-Ct C7-C¶ and C5-N bonds, respectively.
Pro
vents (see Fig. 4). However, the conformation of molecule A is very similar to that proposed (7) for the 1:2 complexes of this type, in which the prolyl carbonyls and the glycyl carbonyls point to the opposite sides of the peptide ring. The sandwich is also stabilized by N-H'0Obonds between the molecules B and A. This sandwich type of cation entrapment is different from the earlier observations in the crystal structures (5, 6, 21-23), in which the cation is associated with a single peptide. The peptide-calcium sandwich has, in addition, swater molecules on one side and a perchlorate anion on the other. The water molecules are involved in O-H ..O hydrogen bonds with the prolyl car-
bonyls of molecules A, resulting in-the formation of a-hydrogenbonded ring, while the perchlorate takes part in N-H--O hydrogen bonds with glycyl nitrogens of molecule B. The coun-
C104
B
Ca
A
X'
-I^ jNd
w
FIG. 2. Calcium is sandwiched between the two peptide molecules A and B and has six coordinating glycyl carbonyl oxygen atoms at an average distance of 2.26-A. There are interpeptide N-IF .0 hydrogen bonds stabilizing the sandwich. The sandwich is bound to a perchlorate anion on one side and water molecules forming a hydrogen-bonded ring with the prolyl carbonyl oxygens on the other side. The complex has a vertical threefold axis passing through the center of the peptide rings, the calcium atom, and the perchlorate.
Chemistry:
Kartha et aL
Proc. Natd. Acad. Sci. USA 79 (1982)
4521
90 ea 4)
-90
FIG. 3. View of the peptide sandwich along the threefold axis with the top peptide ring bonds shown in solid black. The octahedral coordination of the glyc] carbonyls to calcium is seen. tenon thus gives added stability to the unexpected conformation observed in molecule B, in which all six carbonyls point to the same side of the peptide ring, with three of the oxygens coordinating to the cation and the other three forming N-H"O
hydrogen bonds with the peptide A itself. A sandwich of this type seems be effective in sequestering the cation from the solvent. However, dependingon the polarity ofthe surrounding medium, the intrasandwich hydrogen bonds could be broken and the peptide sandwich opened, resulting in conformational changes ofmolecule B and the consequent release of the calcium to
ion into the solvent.
Peptide Mg Complex. The magnesium complex, on the other hand, exhibits a 1:1 stoichiometry for the peptide and the ion, the cation octahedrally coordinating to the three glycyl carbonyls of the peptide and three water molecules (Fig. 5 Left). The crystallographic asymmetric unit contains two peptide molecules, which show very similar coordination geometry and conformation. Both the molecules show noncrystallographic but close threefold symmetry (Table 4), with the peptide conformation being very similar to that of molecule A of the Ca complex. Puckering of the prolines, however, differs in the two molecules. It is seen from Fig. 5 Right that the peptides, the cations, and the water molecules stack to form infinite column along the crystallographic 21 screw axis. The perchlorate anions are located between these columns. The average coordination distance of the Mg2+ and the water oxygens is 2.11 A, whereas that between the cation and the peptide oxygens (2.03 A) is significantly shorter. This difference in coordination distance may be indicative of the greater basicity of the peptide carbonyl. Homologues of (PG)3. NMR studies (24) on cyclo(-L-ProGly-)2 show that the backbone of the peptide is made up of trans-cis-trasw-cis peptides like most other cyclic tetrapeptides. Studies (25) on cyclo(-L-Pro-Gly-)4 show that in chloroform it takes up a C4 symmetric conformation stabilized by y-turns and
'(85,
-90
-181)
90 4,
180
degrees
FIG. 4. plot of molecule A, molecule B, and the 3-y-turn structure. Because all three molecules have threefold symmetry the conformation of each molecule can be representedjust by two points or one vector. The points denoted P and G represent the conformations at the proline and glycine residues, respectively. The coordinates are given in parentheses. The peptide units containing prolines have nearly the same orientation with respect to the threefold axis in all three cases. Proline restricts the rotation, but the 4i values of molecule A and molecule B show a nearly 1700 difference. At glycine residues is nearly the same but 4,values show large differences. The conformation of the 3-rturn structure may be visualized as being midway between the structures of molecules A and B. 4,,
the peptide bonds are trans but the molecule has deviated from C4 symmetry. The rubidium ion has a distorted octahedral environment and is coordinated to four glycyl carbonyl oxygens of one peptide, another glycyl carbonyl of a symmetry-related peptide, and a water oxygen atom. A magnesium complex of cyclo(-Gly-L-Pro-L-Pro-)2 has also been reported (27) recently; it is a discrete sandwich complex with approximately twofold symmetry relating the two peptides of the sandwiches.
an
is made up of all trans peptide units. Crystal structure investigations (26) on the rubidium complex show that here also all
Table 3. Conformation angles (in degrees) of the (PG)3 molecules in the calcium complex Molecule
A B
Residue Pro -64 85 Gly Pro Gly
Xe
144 -175 -7 179 -177
-68 -24 177 -84 -157 -170
Xi
X2
Xs
X4
20 -27 -34 -8
6 -28
39 -34
17
We thank Prof. EMkan Blout for starting our interest in this molecule and Dr. Jake Bello for valuable discussions. This work was supported by U.S. Public Health Service Grant GM-22490 and by the New York State Department of Health.
Table 4. Conformation angles (in degrees) of the (PG)3 molecule in the magnesium complex Mole- Resic Xo XI X2 X3 X cule dueno. Residue 4, 4 A 1 Pro -63 142 -174 4 9 -18 20-13 2 79 -171 -173 Gly 7 -2 -5 3 Pro -57 140 179 9 -9 7 7 -173 -179 4 Gly 5 Pro -64 149 -172 -3 18 -28 25 -13 6 83 172 -176 Gly B
1 2 3 4 5 6
Average Average
Gly Pro Gly
-63 144-171 3-21 92 172 -175 -59 141 -178 18 -36 82 -175 -179 -55 139 -178 9 -29 86 174 177
Pro
-60
Gly
84
Pro
Gly Pro
143 -176 180 -178
32-29
15
40 -30
5
38 -32
15
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Chemistry:
Kartha et al.
Proc. NatL Acad. Sci. USA 79 (1982)
FIG. 5. (Left) View of the peptide-magnesiu. complex indicating the near threefold symmetry. The Mg2" ions are octahedrally coordinated to the three glycyl carbonyls of the peptide and three water molecules (W). (Right) Stacking of the peptide molecules (molecule B) along the b axis. Between the two peptide molecules related by the crystallographic twofold screw axis, in addition to Mg2", there is also a layer of water molecules (W4-s). The other molecule is stacked in an almost identical fashion.
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14. Kartha, G., Ambady, C. & Shankar, P. V. (1974) Nature (London) 247, 204-205. 15. Sobel, H. M., Jamn, S. C., Sakore, T. D. & Nordman, C. E. (1971) Nature (London) New Blot 231, 200-205. 16. Groth, P. (1973) Acta Chem. Scand. 24, 780-790. 17. litaka, Y., Nakamura, H., Tadaka, K. & Takita, T. (1974) Acta Crystallogr. Sect. B 30, 2817-2825. 18. Titlestad, R., Groth, P., Dale, J. & Ali, M. Y. (1973) C/em. Commun., 346-347. 19. Karle, I. L. (1974)J. Am. Chem. Soc. 96, 4000-4006. 20. IUPAG-IUB Commission on Biochemical Nomenclature (1970) J. Mot Biot 57, 1-17. 21. Karle, I. L. (1974) Bioch/emstry 13, 2155-2162. 22. Hamilton, J. A., Steinrauf, L. K. & Braden, B. (1975) Biochem. Biophys. Res. Commurt. 64, 151-156. 23. Kilbourn, B. T., Dunitz, J. D., Pioda, L. A. R. & Simon, W. (1967) J. Mot BotL 30, 559-563. 24. Deber, C. M., Fossel, E. T. & Blout, E. R. (1974)J. Am. Chem. Soc. 96, 4015-4017. 25. Madison, V., Deber, C. M. & Blout, E. R. (1977)J. Am. Chern. Soc. 99, 4788-4798. 26. Chiu, Y. H., Brown, L. D. & Lipscomb, W'. N. (1977) J. Am Chem. Soc. 99, 4799-4803. 27. Karle, I. L. & Karle, J. (1981) Proc Natt Acad. Sc. USA 78, 681-685.
