Characterization at Atomic Resolution of Peptide Helical Structures E. BENEDETTI,’ B. DI BLASIO,’ V. PAVONE,’ C. PEDONE,’ C. TONIOLO,’ and M. CRISMA’
Biocrystallography Centre, CNR, Department of Chemistry, University of Napoli; and ’Biopolymer Research Centre, CNR, Department of Organic Chemistry, University of Padova, Italy
A survey of literature for the various types of helices experimentally observed in highresolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the a-helix, the 310-helix, the fully extended conformation ( 25-helix) and the 6-bend ribbon spiral. For each of these structures the characteristic $ conformational parameters, n , the number of residues per turn, h , the height per residues a n d p , the pitch of the helix are described. #J,
RESULTS AND DISCUSSION
The current widespread use of computer programs in molecular displays as well a s other computer packages for calculating peptide structures brings forth the knowledge of precise a n d accurate geometrical and conformational parameters for the various types of secondary structures explorable by a polypeptide chain. We have carried out a general survey of the various types of helices experimentally observed in high-resolution (better than 1 A ) single crystal xray diffraction analyses of peptides in order t o determine the mean conformational and helical parameters for these secondary structures. T h e results of this study have also provided valuable information on hydrogen bonding, helix aggregation, and packing. Beside the classical a-helix and the less common 310-helix, other two types of peptide helical structures have been characterized, namely the fully extended conformation ( 25-helix)2 and the P-bend ribbon ~ t r u c t u r eEach . ~ of these helices is stabilized by a different scheme of intramolecular hydrogen bonds, involving the donor N-H groups and the acceptor C =0 groups along the polypeptide backbone chain.
The &-Helix and the 3,,,-Helix
Biopolymers, Vol. 32, 453-456 (1992) 0 1992 John Wiley & Sons, Inc.
These helices are stabilized by 1 + 5 and 1 4- 4 intramolecular hydrogen bonds, respectively, giving rise to ring structures containing 13 and 10 atoms ( CI3a n d Clo forms) ! In our statistical analysis we have taken into consideration 54 independent helical molecules, whose crystal structures were determined with a sufficient degree of accuracy (many of them solved in our laboratories). Twenty-two of these molecules showed a t least four consecutive residues in the ahelical conformation, while 32 of them showed at least four consecutive residues in the 310-helical conformation. Out of 408 residues, 258 were found t o be part of helical segments. T h e number of residues in each peptide was comprised between 4 and 16. In Table I the averaged conformational parameters ( d and J / ) observed in peptides for the two types of helices are listed together with the values of various “ideal” structures 5-7 and those recently derived from protein structures.’ The experimentally observed parameters for the 310- and &-helices are significantly different from those of the ideal structures. T h e differences observed result in a greater outward tilt of the carbonyl groups from the helix axis, as shown in Figure 1. 453
BENEDETTI ET AL.
Table I Conformational and Helical Parameters for Right-Handed a- and Bl0-Helices a-Helix
Proteins Ref. 8
Proteins Ref. 8
-63 -42 1.56 3.63 5.67
-62 -41 1.52 3.54 5.40
-48 -57 1.51 3.65 5.57
-67 -44 1.42 3.67 5.2
-57 -47 1.53 3.59 5.5
-57 -30 1.94 3.24 6.29
-71 -18 1.81 3.20 5.80
-74 -4 2.00 3.00 6.00
-49 -26 1.93 3.0 5.8
The mean #,I)angles for right-handed a-helices found in peptides and proteins are very close; however, for 310-helicesthe $,# angles observed in peptides and proteins differ substantially. It should be noted that in the case of peptides the 310-helicalsegments are longer than those observed in proteins: the average length of 310-helices in peptides is 4.9 residues, while in proteins these helical segments have a mean length of 3.3 residues. In addition, the peptide 310-helicesare much more regular, having a smaller spread of the conformational angles. In Table I the calculated averaged values of the helical parameters h , height per residue, n , number of residues per turn, and p, pitch of the helix, are also listed and compared with those of the ideal he lice^^-^ and those derived from protein crystal structures.* In particular, it is worth noting that the experimentally observed nonintegral number of residues per turn for the 310-helixin both peptides and proteins does not line up side chains, thereby inducing a slightly staggered, energetically more favorable disposition. The present analysis of peptide crystal structures has also allowed us to derive the following preferences and trends: 1. The minimum peptide main-chain length for a-helix formation corresponds to 7 residues. Slo-Helices require a smaller number of residues (as low as four residues, when C","-dialkylated residues are present in the sequence). 2. Usually, two or three turns of helix are seen; quite often, a-helical segments terminate at both ends with few residues in the 310-helical conformation. 3. Helical molecules, regardless of their type, pack one on top of the other by forming intermolecular hydrogen bonds between the Nterminal N-H donor group of one molecule and the C-terminal C=O acceptor group of a symmetry-related molecule ( o r another in-
dependent molecule in the unit cell). Usually, the formation of long rows of hydrogenbonded molecules is seen. These rows then pack with each other in an antiparallel or in a parallel fashion by forming an hexagonal array (see also Ref. 9 ) . The Fully Extended Peptide Conformation (2,-Helix)
We have recently carried out conformational energy computations and crystal-state structural analyses of a variety of derivatives and short peptides from the symmetrically disubstituted C "3"-dialkylglycines Deg (diethylglycine) , Dpg (di-n-propyl-glycine), D9g (diphenylglycine) , and Db,g (dibenzylglycine) .2 For these residues the conformational space explorable is severely restricted, and the stabilities of the 310- and a-helical structures decrease by lengthening the number of side-chain carbon atoms relative to the uncommon fully extended, intramolecularly hydrogen-bonded conformation ( 25-helix). Actually, in 19 out of 21 residues of Deg and Dpg derivatives and homopeptides (to the pentamer), examined by x-ray diffraction techniques, the Z5helical conformation has been found. An example of this structure is given in Figure 2, where the molecular model of the fully extended pentapeptide Tfa- (Deg),-OtBu is reported. From our study we have been able to characterize at atomic resolution this novel structure, which is stabilized by consecutive 2 --* 2 ( C5 type) hydrogen bonds along the chain.4 The mean conformational and helical parameters for the Z5-helix are # = # = w = =18O0, n = 2.06, h = 3.58 A, a n d p = 7.36 A. Interestingly, the mean value ofthe N-C"-C' ( 7 ) bond angle is as low as 103". The &Bend Ribbon Spiral
This helical structure can be described as having approximately a 310-helicalfold of the peptide chain. However, it is stabilized by half the number of 1 +
CHARACTERIZATION A T ATOMIC RESOLUTION
Figure 1. The segment of a-helix seen in the central portion of the - (Aib-L-Ala),- structure ( A ) and the segment of 310-helixseen in the - (Aib)lo-structure ( B ) . ( C ) The models of the a-helix (left) and 310-helix(right), as calculated with the average values given in Table I, are illustrated perpendicularly (top) and along (bottom) the helix axis, respectively. Intramolecular hydrogen bonds are indicated as dashed lines.
BENEDETTI ET AL.
Figure 2. The molecular structure of Tfa-(Deg),-OtBu: a model for the Z5-helix.Terminal blocking groups (Tfa: trifluoroacetyl; OtBu: tert-butoxy ) have been omitted for clarity. Intramolecular hydrogen bonds are indicated as dashed lines. 4 ( CI0 type) intramolecular hydrogen bonds, since
along the chain a N-alkylated amino acid residue is sequentially alternating with an amino acid. This structure, which can be considered a subtype of the 310-helix, has been characterized by solving the crystal structures of two peptides of the series pBrBz- ( Aib-L-Pro) n-Aib-OMe with n = 3,4.1° The
molecular structure of the nonapeptide is given in Figure 3. The @-bendribbon structure has been characterized in the structure of Ref. 3. It is relevant to the development of structural models for peptaibol antibiotics l1 and for the numerous ( Xxx-L-Pro), segments found in proteins.' As far as the (Aib-LPro) repeating unit is concerned, the following conformational parameters-& = -54", = -40", o1 = -175", = -78", q2 = -lo", o2 = -169" are obtained. The resulting helix is described by the helical parameters n = 3.43, h = 2.06 A, andp = 7.0 A.
Figure 3. The molecular structure of pBrBz-Aib- ( LPro-Aib),-OMe: a model for the &bend ribbon spiral. Terminal blocking groups (pBrBz: parabromobenzoyl; OMe: methoxy ) have been omitted for clarity. Intramolecular hydrogen bonds are indicated as dashed lines.
1. Toniolo, C. & Benedetti, E. (1991) Trends Biochem. Sci., in press. 2. Toniolo, C. & Benedetti, E. (1991) in Molecular Conformation and Biological Interactions: G. N. Ramachandran Festschrift, Balaram, P. & Ramaseshan, S., Eds., Indian Institute of Science, Bangalore, in press. 3. Karle, I., Flippen-Anderson, J., Sukumar, M. & Balaram, P. (1987) Proc. Natl. Acad. Sci. USA 84,5087509 1. 4. Toniolo, C. ( 1980) CRC Crit. Rev. Biochem. 9, 1-44. 5. Pauling, L., Corey, R. B. & Branson, H. R. (1951) Proc. Natl. Acad. Sci. USA 37,205-211. 6. Perutz, M. F. (1951) Nature (London) 167,10531054. 7. Arnott, S. & Wonacott, A. J. (1966) J . Mol. Biol. 21, 371-383. 8. Barlow, D. J. & Thornton, J. M. (1988) J. Mol. Biol. 201,601-619. 9. Karle, I. L. & Balaram, P. (1990) Biochemistry 29, 6747-6756. 10. Benedetti, E., Di Blasio, B., Pavone, V., Pedone, C., Crisma, M., Anzolin, M. & Toniolo, C. (1991) in Peptides: Chemistry and Biology, Smith, J. A. & Rivier, J. E., Eds., ESCOM, Leiden, The Netherland, in press. 11. Benedetti, E., Bavoso, A., Di Blasio, B., Pavone, V., Pedone, C., Toniolo, C. & Bonora, G. M. ( 1982) Proc. Natl. Acad. Sci. USA 79,7951-7954.
Received J u n e 10, 1991 Accepted August 7, I991