Proc. Natl. Acad. Sci. USA Vol. 83, pp. 9284-9288, December 1986 Chemistry Parallel packing of a-helices in crystals of the zervamicin IIA analog Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe 2H20 (x-ray structure analysis/hydrogen bond/helix curvature/ionophore/membrane channel) ISABELLA L. KARLE*, MUPPALLA SUKUMARt, AND PADMANABHAN BALARAMt *Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375-5000; and tMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India Contributed by Isabella L. Karle, September 8, 1986 ABSTRACT An apolar synthetic analog of the first 10 crystals were grown from dimethyl sulfoxide/H20 in the residues at the NH2-terminal end of zervamicin HA crystallizes form of triangular prisms. X-ray diffraction data were mea- in the triclinic space group P1 with cell dimensions a = 10.206 + sured from a crystal that was 0.10 mm thick and 0.30 mm on 0.002 A, b = 12.244 + 0.002 A, c = 15.049 ± 0.002 i, a = 93.94 each side of the triangular face. The crystal was sealed in a ± 0.010, /8 = 95.10 ± 0.010, y = 104.56 _ 0.01°, Z = 1, capillary with a small amount ofmother liquor. The data were C6oH97N1103-2H2O. Despitetherelativelyfew a-aminoisobutyric collected with a four-circle automated diffractometer using acid residues, the peptide maintains a helical form. The first Cu Ka radiation and a graphite monochromator (A = 1.54178 intrahelical hydrogen bond is of the 310 type between N(3) and A). The 0-20 scan technique was used with a 2.00 scan, 0(0), followed by five a-helix-type hydrogen bonds. Solution 1H 15'/min scan rate, and 2ma,,x = 1150, for a total of 5294 NMR studies in chloroform also favor a helical conformation, independent reflections and 4378 reflections with intensities with seven solvent-shielded NH groups. Continuous columns are >3a(F) to a resolution of0.91 A. Three reflections monitored formed by head-to-tail hydrogen bonds between the helical after every 60 measurements remained constant within 3% molecules along the helix axis. The absence of polar side chains during the data collection. Lorentz, polarization, and absorp- precludes any lateral hydrogen bonds. Since the peptide crystal- tion corrections were applied to the data. The space group is lizes with one molecule in a trilinic space group, aggregation of P1 with a = 10.206 ± 0.002 A, b. = 12.244 ± 0.002 A, c = the helical columns must necessarily be parallel rather than 15.049 ± 0.002 A, a = 93.94 + 0.010, / = 95.10 ± 0.010, fly = antiparallel. The packdng of the columns is rather inefficient, as 104.56 ± 0.010, V = 1804.9 + 0.5 A3, Z = 1, calculated density indicated by very few good van der Waals' contacts and the = 1.119 g/cm3 based on a molecular weight of 1180.52 + occurrence of voids between the molecules. 36.03 for C6oH97N1O113-2H2O, and one formula unit per cell. The structure was solved by direct phase determination A number of naturally occurring peptides containing a- using the random-tangent formula procedure in the aminoisobutyric acid (Aib) residues form voltage-dependent SHELXTL computer programt. Initially, the positions of 36 channels across lipid bilayer membranes. Transmembrane atoms, mainly in the backbone, were found. The positions of channels are likely to be formed by close association of the the remainder ofthe C, N, and 0 atoms were found by means peptide helices in the lipid bilayers, as already indicated by of partial structure development. Eight of the nine H atoms the structure of alamethicin (1). Individual 310- or a-helices on amide groups were found in difference maps after partial have an inner pore too small for passage of ions. Therefore, least-squares refinement, and subsequently their coordinates channel formation requires the aggregation ofpeptide helices and isotropic thermal factors were refined by least-squares. (2). The role of specific side chains in promoting helix Hydrogen atoms for the two water molecules were not found. association is being studied by means of synthetic analogs. Although peaks for many other H atoms also were found in The present study has begun with apolar analogs of zerva- difference maps, idealized calculated positions were used for micin IIA: all the H atoms on C atoms. Least-squares refinement with anisotropic thermal factors for the C, N, and 0 atoms; 10 Ac-Trp-Ile-Gln-Aib-Ile-Thr-Aib-Leu-Aib-Hyp-5 isotropic thermal factors for eight amide H atoms; and 88 H 15 atoms on C atoms kept fixed in idealized positions yielded an -Gln-Aib-Hyp-Aib-Pro-Phe-ol. agreement factor of R = 5.61% for the 4378 reflections measured >3a(F). Fractional coordinates for the C, N, and (Hyp is 4-hydroxyproline; Phe-ol is phenylalaninol.) The o atoms and the amide H atoms are listed in Table 1. Bond decapeptide whose structure is reported in this paper is the lengths and angles are shown in Tables 2 and 3.§ apolar analog of the first 10 residues in which Gln-3, Thr-6, and Hyp-10 have been replaced with Ala, Val, and Pro, RESULTS respectively; the NH2 terminus has been blocked with t-butoxycarbonyl (Boc) rather than acetyl; and the COOH Conformation of Molecule. The backbone of Boc-Trp-Ile- terminus has been esterified with methanol: -Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe, in which all the res- idues are nonpolar and hydrophobic, folds into an a-helix for 5 10 most of its length. The helix is initiated by the Trp residue at Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe. the NH2 terminus. There is a helix reversal at residue Aib-9 EXPERIMENTAL PROCEDURE Abbreviations: Aib, a-aminoisobutyric acid; Boc, t-butoxycarbonyl; Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe was Phe-ol, phenylalaninol; OBut, t-butoxy. by conventional solution-phase procedures, and tSheldrick, G. M. (1981) SHELXTL, An Integrated System for synthesized Solving, Reffning and Displaying Crystal Structures from Diffrac- tion Data (Univ. of Gottingen, F.R.G.). The publication costs of this article were defrayed in part by page charge §Supplementary material consisting of observed and calculated payment. This article must therefore be hereby marked "advertisement" structure factors, anisotropic thermal factors, and coordinates for in accordance with 18 U.S.C. §1734 solely to indicate this fact. the H atoms are available from I.L.K. 9284 Downloaded by guest on October 2, 2021 Chemistry: Karle et al. Proc. Natl. Acad. Sci. USA 83 (1986) 9285 Table 1. Atomic coordinates (x 104) and thermal factors (A2 x 103) Mean value (standard deviation) Mean value (standard deviation) Atomt x y z Uqt Atom x y z Ueqt C(OBu9)-5 10592 (8) 9053 (8) 4699 (5) 148 (4) C,B(6) 9482 (7) 6661 (5) -1180 (5) 102 (3) C(OBu')-4 10580 (10) 7379 (10) 5555 (5) 173 (6) Cyl(6) 9211 (10) 7813 (6) -1072 (5) 145 (4) C(OBu9-3 10443 (9) 9193 (11) 6351 (5) 195 (6) Cy2(6) 10601 (8) 6614 (7) -1793 (5) 141 (4) C(OBu')-2 10069 (9) 8425 (8) 5486 (5) 125 (4) N(7) 8571 (4) 3930 (4) -1206 (3) 68 (2) O(OBu' 8591 (4) 8129 (5) 5390 (3) 58 (2) Ca(7) 8756 (6) 2796 (5) -1429 (4) 77 (3) C'(O) 7861 (6) 7516 (5) 4696 (4) 87 (3) C'(7) 7540 (7) 2084 (5) -2064 (3) 85 (3) 0(0) 8259 (4) 6898 (4) 4159 (2) 94 (2) 0(7) 7715 (5) 1388 (4) -2644 (3) 119 (2) N(1) 6549 (5) 7583 (4) 4599 (3) 74 (2) Cp1l(7) 8818 (7) 2234 (5) -551 (3) 89 (3) Ca(1) 5545 (5) 6899 (4) 3902 (3) 63 (2) C,82(7) 10107 (7) 2879 (6) -1830 (4) 109 (3) C'(1) 5921 (5) 7064 (4) 2962 (3) 58 (2) N(8) 6306 (5) 2199 (3) -1931 (2) 69 (2) 0(1) 5552 (3) 6294 (3) 2363 (2) 65 (1) Ca(8) 5084 (7) 1536 (4) -2489 (3) 77 (2) Cp(1) 4122 (5) 7069 (4) 3998 (3) 66 (2) C'(8) 4975 (7) 1915 (5) -3436 (4) 83 (3) Cy(1) 4008 (5) 8236 (4) 3885 (3) 63 (2) 0(8) 4382 (5) 1234 (3) -4066 (2) 103 (2) C51(1) 4142 (6) 9102 (5) 4529 (4) 78 (2) Cl3(8) 3818 (7) 1529 (5) -2039 (3) 81 (2) NEl(1) 3992 (5) 10050 (4) 4163 (3) 85 (2) Cy(8) 3648 (7) 877 (5) -1200 (4) 88 (2) CE2(1) 3692 (6) 9816 (4) 3257 (4) 70 (2) C81(8) 2431 (9) lq1 (8) -767 (5) 146 (4) CC2(1) 3382 (7) 10503 (5) 2620 (5) 100 (3) C82(8) 3516 (9) -364 (5) -1447 (5) 136 (4) C-12(1) 3120 (9) 10040 (6) 1755 (5) 116 (4) N(9) 5498 (6) 3021 (4) -3512 (3) 89 (2) CQ3(1) 3100 (8) 8902 (7) 1505 (4) 110 (4) Ca(9) 5637 (8) 3462 (5) -4398 (3) 98 (3) CE3(1) 3388 (7) 8225 (5) 2159 (4) 85 (3) C'(9) 4312 (7) 3068 (4) -5024 (3) 84 (3) C52(1) 3697 (5) 8683 (4) 3051 (4) 67 (2) 0(9) 4313 (4) 2625 (3) -5788 (2) 89 (2) N(2) 6704 (4) 8085 (3) 2818 (2) 56 (1) Cpl1(9) 6061 (11) 4751 (6) -4249 (4) 146 (4) Ca(2) 7076 (5) 8282 (4) 1919 (3) 56 (2) C,B2(9) 6756 (8) 3053 (8) -4805 (4) 139 (4) C'2 7916 (5) 7480 (4) 1614 (3) 59 (2) N(10) 3118 (8) 3204 (5) -4776 (4) 104 (3) 0(2) 7858 (4) 7189 (3) 802 (2) 69 (1) Ca(10) 1916 (8) 2787 (7) -5427 (5) 117 (4) C,(2) 7773 (6) 9541 (4) 1855 (3) 72 (2) C'(10) 2044 (7) 3398 (5) -6267 (5) 97 (3) Cyl(2) 7888 (7) 9801 (5) 886 (4) 97 (3) 0(10) 2573 (6) 4355 (3) -6286 (3) 126 (2) Cy2(2) 9140 (7) 9916 (5) 2393 (4) 100 (3) C/3(10) 776 (11) 3062 (12) -4927 (7) 228 (7) C81(2) 6573 (9) 9580 (6) 275 (4) 129 (4) CB(10) 1486 (16) 3673 (16) -4093 (7) 294 (9) N(3) 8715 (4) 7143 (3) 2225 (3) 57 (2) C8(10) 2850 (12) 3650 (10) -3898 (5) 184 (6) Ca(3) 9511 (5) 6381 (4) 1969 (3) 64 (2) O(OMe) 1403 (5) 2698 (4) -6982 (4) 111 (2) C'(3) 8629 (5) 5241 (4) 1533 (3) 58 (2) C(OMe) 1369 (9) 3185 (7) -7822 (5) 132 (4) 0(3) 8908 (4) 4756 (3) 864 (2) 71 (1) W(1)§ 7343 (7) 296 (5) 4057 (4) 177 (3) CB(3) 10432 (6) 6226 (5) 2765 (4) 84 (2) W(2)§ 7102 (11) 9915 (8) 5893 (5) 258 (6) N(4) 7529 (4) 4788 (3) 1952 (2) 59 (2) H(1) 6313 (42) 8016 (33) 4847 (25) 50 (11)¶ Ca(4) 6536 (5) 3712 (4) 1617 (3) 62 (2) H(2) 6923 (41) 8745 (32) 3205 (25) 59 (11)$ C'(4) 5974 (5) 3812 (4) 641 (3)