J. Mol. Biol. (1990) 214, 633-635

The Longest, Regular Polypeptide Atomic Resolution Vincenzo

Pavone, Benedetto

Di Blasio, Antonello Carlo Pedone

3,, Helix at Santini,

Ettore Benedetti

Department of Chemistry University of Naples 80134 Naples, Italy

Claudio

Toni010

and Marco Crisma

Biopolymer Research Center, C.N.R. Department of Organic Chemistry University of Padova, 35131 Padova, Italy (Received 1 February

1990; accepted 4 April

1990)

-dimethylated glycyl residue A synthetic, terminally blocked homodecapeptide from the C”, OL a-aminoisobutyric acid has been analyzed by single-crystal X-ray diffraction and the structure refined to R = 0073. The compound crystallizes as a perfect 3,, helix, stabilized by eight consecutive intramolecular N-H . O=C hydrogen bonds. This is the first observation at atomic resolution of a regular polypeptide 3ro helix as long as three complete turns.

gem-methyl groups linked to the a-carbon, has been documented by X-ray diffraction studies of suitable model peptides, and of the membrane-active, channel-forming, “quasi’‘-ionophoric, peptaibol antibiotics and numerous fragments and analogs (for recent reviews, see Prasad & Balaram, 1984; 1988). Toniolo et al., 1986; Toniolo & Benedetti, Here, we describe the crystal-state structure of the synthetic, terminally blocked homodecapeptide from Aib, pBrBz-(Aib),,-OtBu (pBrBz, parabromobenzoyl; OtBu, t-butoxy), which represents the longest: regular 3ro helix solved to date by X-ray diffraction. This investigation allowed us to characterize this important peptide ordered secondary structure in great detail. We incorporated the PBrBz group at the N terminus toI help solve the phase problem in the X-ray diffraction analysis, since it possesses a suitable heavy--atom (W The decapeptide was synthesized by the 51(4H)oxazolone method (Leplawy et al., 1960) from pBrBz-(Aib),-OH and H-(Aib),-OtBu and purified by crystallization from hot acetonitrile (m.p. 322 to 323°C). Crystals were grown from a methanol solution by slow evaporation. A single crystal measuring 0.1 mm x 62 mm x 0.1 mm was used to collect

Beside the a-helix, the only other principal helical structure that occurs to any great extent in globular proteins is the 3r0 helix, with an ideal 3.0 residue repeat (instead of 3.6) and an i +- i+ 3 intramolecular C=O . . . H-N hydrogen bonding pattern (instead of i t i+4: Barlow & Thornton, 1988). Its backbone torsion angles are approximately cp = -6O”, ti = -3O”, within the same energy minimum as the a-helix (cp = -555”, 9 = - 45”). However, for a long periodic structure formed by C”-monosubstituted tl amino acid residues, the 310 helix is significantly less stable than the a-helix. Therefore, it is very unlikely that long stretches of the 3,0 helix would be observed. In a recent survey of all helices formed in 57 of the known protein crystal structures, Barlow & Thornton (1988) showed that: (1) 3.4% of the residues are involved in 310 helices; (2) the majority of 310 helices are very short (mean length = 3.3 residues); and (3) the 310 helices are generally irregular, in that they have a larger radius and a smaller pitch (mean value for cp, I) = -70”; - 18”). More recently, the ability of the C”,“-disubstituted a amino acid residue Aib (c+aminoisobutyric acid or C”‘“-dimethylglycine) to promote the onset of 31,, helices, due to steric interactions of the 0022-2836/90/15063343

$03.00/O

633

0 1990 Academic Press Limited

V. Pavone et al.

634

Figure 1. Molecular structure of pBrBz-(Aib),,-OtBu. In this Figure, the right-handed The 8 intramolecular hydrogen bonds are represented by broken lines.

3,, helical molecule is shown.

& 31.2”, respectively, very close to the ideal case. All eight intramolecular 1 +- 4 hydrogen bonds, appropriate for a 310 helix, are found. The range of observed N 0 distances is 2.91 to 3.21 A (mean value, . 0 and 3.04 A), while those of N-H N O=C angles are 149 to 173” (mean value, 159”) and 125 to 136” (mean value, 130”), respectively (Ramakrishnan & Prasad, 1971; Taylor et al., 1984), The deviations of the o angles from the ideal value of the tram planar peptide unit’ (180”) are very small, the average value being 3.9”. In this helix, on the average, one peptide group is carried into the next, to which it is directly chemically linked by a rotation of 112” and an axial translation of 1.96 A. The pitch is 6.29 A, and there are 3.21 residues per turn. The separation of the methyl groups of one residue from the methyl groups on both adjacent turns of the helix is always greater than 4.2 A. The helix extends from residue 1 to residue 9 (3 complete turns), the sign of the torsion angle of the Cterminal Aib residue being opposite to those of the preceding residues (a common feature of the cryst’alstate structures of the 3,, helical Aib-rich peptides). One water molecule eocrystallizes in the unit cell. linking toget,her symmetry-related decapeptide molecules along the a-c direction by the formation of two intermolecular hydrogen bonds, with the N-H group of residue 1 and the C=O group of residue 9: respectively (the corresponding N . . 0, and 0 . 0, distances are 2.97 A and 2-88 A, respectively). In the a-c direction, the resulting long columns of peptide molecules linked by the water

the diffraction data. Space group C2/c (monoa = 43.901(2) A, clinic); b = 9.289(l) A, and 2 = 8; v= c = 34+746(3) 8; B = 114.69(3)“; 12874.2 A3; 0, = 1.143 g cme3; CuKa radiation, /z = 1.5418 A (1 A = 91 nm). Intensity data collection was carried out on an Enraf-Nonius CAD4 diffractometer in the 8 range 2 to 65”. A total of 11,933 independent reflections were measured, 6819 of which had a net intensity I 2 3.00(I) and were considered “observed”. The structure was solved by Patterson peak analysis, which revealed the position of the bromine atom. The positions of the remaining atoms were derived from subsequent Fourier syntheses. Refinement was carried out, using the SDP package (Structure Determination Programs) and full-matrix least-squares procedures, with weights w equal to l/a(Ff). Hvdrogen atoms were introduced in their stereochemically expected positions with isotropic thermal factors equal to the equivalent’ B factor of the atom to which each of them was bonded. The final R and R, factors for the observed reflections are 0073 and 0.076, respectively. A stereo view of the decapeptide molecule is illustrated by Figure 1. The torsion angles that define the backbone conformation (IUPAC-IUB Commission, 1970) are given in Table 1. Each molecule, having no chiral atoms, crystallizes with retention of the center of symmetry; thus, molecules of both hand are found in each unit cell. The peptide assumes a regular 3,* helical conformation with mean cp and II/ values of + 54.1” and

Table Backbone

torsion

angles

(cp, $, and o) for pBrBz-(Aib),,-e)tBu Residue number

Torsion angle (deg.) cp(C-, -N,-C;-C:-) $(Ni-C;-C;-Ni+,) o(Cq-C;-N,+l-C~;,)

1

2

3

-52 -35 -178

-48 -33 -177

-49

-60

-42 -173

-28

4

-177

5

6

-53 -36 -174

-21 -178

-59

7

8

-56 -24 -178

-36 -178

-49

9 -61 -26 -172

10 47

-

Communications

molecule, pack together in an antiparallel fashion with only van der Waals interactions. In this work, we have characterized a regular 3,0 helical structure, a relevant feature of globular proteins and peptaibol antibiotics, in greater detail than was possible previously with shorter (Aib), homopeptides (Bavoso et al., 1986; Benedetti et al., 1982; Shamala et al., 1978). It is also our contention that the present results represent a decisive proof in favor of this ordered secondary structure as the preferred conformation of poly(Aib),, and that main-chain length may not be an overriding parameter in directing the type of helical folding in this homopolymer References Barlow, D. J. & Thornton, J. M. (1988). J. Mol. Biol. 201, 601-619. Bavoso, A., Benedetti, E.; Di Blasio, B., Pavone, V.,

Edited

635

Pedone, C., Toniolo, C. & Bonora, G. M. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 1988-1992. Benedetti, E., Bavoso, A., Di Blasio, B., Pavone, V., Pedone, C., Crisma, M., Bonora, G. M. & Toniolo, C. (1982). J. Amer. Chem. Sot. 104, 2437-2444. IUPAC-IUB Commission on Biochemical Nomenclature (1970). J. &foZ. Biol. 52, 1-17. Leplawy, ‘M. T., Jones; D. S., Kenner, G. W. & Sheppard, R. C. (1960). Tetrahedron, 11, 39-51. Prasad, B. V. V. & Balaram, P. (1984). C.R.C. Crit. Rev.

Biochem. 16, 307-348. Ramakrishnan,

C. & Prasad,

N. (1971).

Int. J. Protein

Res. 3, 209-231. Shamala,

N., Nagaraj,

R. 8: Balaram,

P. (1978). J. Chem.

SOL, Chem. Commun. 996-997. R., Kennard, 0. & Versichel, W. (1984). Ada Crystallogr. sect. B, 40, 280-288. Toniolo, C. & Benedetti, E. (1988). ISI Atlas of Science: Biochemistry, 1, 225-230.

Taylor,

Toniolo,

C., Benedetti,

E. & Pedone,

Chim. Ital. 116, 355-359.

by R. Huber

C. (1986).

Gazz.

The longest, regular polypeptide 3(10) helix at atomic resolution.

A synthetic, terminally blocked homodecapeptide from the C alpha, alpha-dimethylated glycyl residue alpha-aminoisobutyric acid has been analyzed by si...
256KB Sizes 0 Downloads 0 Views