Protein Engineering, Vol. 15, No. 4, 331-335,
April 2002
© 2002 Oxford University Press
Dehydrophenylalanine zippers: strong helix–helix clamping through a network of weak interactions
1 Department of Physics and 2 Bioinformatics Center, Indian Institute of Science, Bangalore-560012, 3 Department of NMR, All India Institute of Medical Sciences, New Delhi-110029 and 4 International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India
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Abstract |
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A decapeptide Boc-L-Ala-(
Phe)4-L-Ala-(Phe)3-Gly-OMe (Peptide I) was synthesized to study the preferred screw sense of consecutive 
,ß-dehydrophenylalanine (Phe) residues. Crystallographic and CD studies suggest that, despite the presence of two L-Ala residues in the sequence, the decapeptide does not have a preferred screw sense. The peptide crystallizes with two conformers per asymmetric unit, one of them a slightly distorted right-handed 310-helix (X) and the other a left-handed 310-helix (Y) with X and Y being antiparallel to each other. An unanticipated and interesting observation is that in the solid state, the two shape-complement molecules self-assemble and interact with an extensive network of C–H···O hydrogen bonds and 
– interactions, directed laterally to the helix axis with amazing regularity. Here, we present an atomic resolution picture of the weak interaction mediated mutual recognition of two secondary structural elements and its possible implication in understanding the specific folding of the hydrophobic core of globular proteins and exploitation in future work on de novo design.
Keywords: aromatic interactions/dehydrophenylalanine/de novo design/310 helix
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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De novo design is a hierarchical approach towards the construction of novel mini-proteins with predetermined three-dimensional structure and defined biological function (Baltzer, 1999
; De Grado et al., 1999). Incorporation of conformation-restricting amino acids into polypeptide sequences is being investigated to design molecules with desired secondary structure (Balaram, 1999). ,ß-Dehydroamino acids are natural, non-coded amino acids which introduce conformational constraints in the peptide backbone and have been incorporated in many bioactive peptide sequences (Jain and Chauhan, 1996). Dehydrophenylalanine (Phe), one of the best studied dehydroamino acids, induces type II ß-bends in short sequences and right-handed, 310-helical conformations in longer peptides. Recently, it was shown that Phe is also a potential residue to introduce long-range interactions to achieve the folding of super secondary structures such as a helical hairpin motif (Ramagopal et al., 2001).
The peptide Boc-L-Ala-(Phe)4-L-Ala-(Phe)3-Gly-OMe (Peptide I) was designed to understand the preferential helical screw sense observed in the consecutive Phe-containing peptides studied in our laboratory and by others (Day et al., 1996; Jain et al., 1997; Ramagopal et al., 1998). The results presented in this paper depict an unanticipated but interesting mutual recognition and association of ambidextrous helices mediated by a network of weak interactions (Desiraju and Steiner, 1999) and highlight its possible exploitation in future work on 2de novo design. It is observed that the two shape-complement 310-helices in the asymmetric unit, a slightly distorted right-handed 310-helix (X) and a left-handed 310-helix (Y), are clamped together by a C–H···O hydrogen-bonded Phe zipper structure. They are further supported by – interactions (Malone et al., 1997; Steiner, 1998). The planar Phe side chains from the adjacent helices stack one above the other, forming `extended phenyl embrace' arrangements at the helix–helix interface. Here we report how molecular recognition mediated by weak interactions can be exploited towards the construction and design of folded super secondary structural elements.
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Materials and methods |
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The decapeptide was synthesized by standard solution-phase methods. Consecutive
Phe residues were introduced using the already reported procedures (Jain et al., 1997). All the reactions were monitored by TLC on precoated silica plates. The decapeptide was eluted as a single peak in an HPLC run on a Waters C18 column (300x3.9 mm i.d.) using a methanol–water gradient and UV detection at 280 nm. The peptide (C77H74O13N10) was fully characterized by mass and 400 MHz 1H NMR spectrometric analysis. X-ray intensity data for Peptide I were collected on a Rigaku-AFC7 diffractometer with Cu K radiation (
= 1.5418 Å) up to a Bragg angle of (2
) 120° using a crystal grown by controlled evaporation of the peptide solution in N,N-dimethylformamide (DMF)–methanol mixture at room temperature. The crystal belongs to the triclinic space group P1 with a = 15.711(1), b = 20.372(1), c =14.987(1) Å, = 98.816(6), ß = 105.539(6), 
= 105.602(6)°, Z = 2, GOF = 1.64 and dc = 1.147 g/cm3. A total of 11 749 unique reflections were collected of which 6411 had |Fo| > 4
|Fo|. The structure was determined using direct methods applying the SHELXS97 computer program. Refinement was carried out using SHELXH97 (Sheldrick, 1997) on |F|2 using all the reflections with anisotropic temperature factors for non-hydrogen atoms. It was observed that the phenyl ring of the Phe2(Y) residue was disordered. During the course of refinement, three DMF [named DMF(S1), DMF(S2) and DMF(S3)] molecules with full occupancy, one disordered methanol with fractional occupancy and a few highly disordered water molecules were located in the difference Fourier map. Solvent atoms having unusually high temperature factors were refined isotropically. All the hydrogen atoms were fixed using stereochemical criteria and were used only for structure factor calculations. The conventional R-factor is 9.95% for 6411 reflections with |Fo| > 4|Fo| and 1963 variables. Circular dichroism (CD) measurements were carried out on a JASCO J720 spectropolarimeter with a data processor attached. A 1 mm pathlength was used. The spectrum was recorded in various solvents such as acetonitrile, dichloromethane, hexafluoro-2-propanol and trifluoroethanol. Chloroform–methanol titrations were also carried out by increasing the concentration of methanol from 0 to 80% and the data were expressed in terms of molar ellipticity.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Molecular conformation
A perspective view of the ambidextrous molecules in the asymmetric unit perpendicular to the two axes of the helices is given in Figure 1. The right-handed 310-helix (X) and left-handed 310-helix (Y) are anti-parallel to each other. The angle between the two helical axes is 
15°.
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The molecule X is characterized by a distorted right-handed 310-helical conformation. The 310-helical structure is maintained between
Phe3 and Phe9, forming two complete helical turns as a result of six consecutive, overlapping type III ß-bends (Table I), stabilized by appropriate 4
1 intramolecular N–H···O hydrogen bonds (Table I). The average main chain dihedral angles for the residues from Phe3 to Phe9 are <
> = –55.9° and <
> = –21.6°. The N-terminus of molecule X is unwounded and the carbonyl oxygen atoms of the Boc(O0') and the first N-terminal residue (O1') do not participate in intramolecular hydrogen bonding. The , values for L-Ala1 and Phe2 are –100.3, 4.8° and 104.0, –5.2°, respectively. As a result of this unusual unwinding, the first four amide NH groups in molecule X [N1(X), N2(X), N3(X) and N4(X)] are free. The carbonyl oxygen of the two solvent molecules DMF(S1) and DMF(S2), which co-crystallize with the peptide molecule, accepts two hydrogen bonds each (N–H···O) and cross-link N1(X) with N3(X) and N2(X) with N4(X), respectively (Figure 2, Table I; see supplementary material for more information).
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The molecule Y is characterized by a left-handed 310-helical conformation (Figure 1
) composed of eight consecutive, overlapping ß-bends stabilized by appropriate 41 intramolecular N–H···O hydrogen bonds (see supplementary material). The first bend, a non-helical type II ß-bend involving the first two residues -L-Ala1-Phe2-, is followed by seven type III' ß-bends. The average main chain dihedral angle for the residues accommodated in the 310-helix are <> = 56.9° and <>= 20.7°. Interestingly, L-Ala6, although an L-chiral residue, assumes dihedral angles ( = 49.9°, = 32.1°) with positive , corresponding to the left-handed -helical region (Table II). The side chain phenyl ring of Phe2 is disordered in the left-handed helix. It is also noteworthy that the conformations of the four `-L-Ala-Phe-' stretches in the different positions and environments of the molecules X and Y are entirely different from one another (Table I).
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The packing of the peptide molecules in the solid state is illustrated in Figures 1 and 2
. The amphipathic solvent molecules, DMF(S1), which cross-links N1(X) with N3(X), and DMF(S2), which cross-links N2(X) with N4(X) by making two hydrogen bonds each, at their polar end, also participate in lateral C–H···O hydrogen bonds with the symmetry-related molecules at their apolar end ( Figure 2 and Table I).
In the crystal, the helices related by translational symmetry are observed as approximate helical rods and staggered by a small amount along the b-axis. A solvent molecule named DMF(S3) mediates the head to tail hydrogen bond (Karle, 1996) by making N2(Y)–H···O1S3, C1S3–H···O0'(Y) hydrogen bonds at its polar end and C3S3–H···O8'(Y) hydrogen bond at its apolar end with a symmetry related molecule (Table I, see figure in the supplementary material). Similarly, there is no direct N–H···O=C head-to-tail hydrogen bond between the two right-handed helices. Rather the C
1(X) from the Phe3 side chain forms a weak hydrogen bond with the free acceptor O9'(X) of the symmetry-related molecule (see figure in supplementary material for details).
A remarkable feature of the present structure is that the two shape-complement helices named X and Y are held together by symmetrically placed aromatic-backbone C–H···O interactions distributed all along the helical axis (Figure 1, Table I). Thus C–H(phenyl)···O(carbonyl) hydrogen bonds (Burley and Petsko, 1988) are observed between C
2(Phe2), C2(Phe5), C2(Phe8) of molecule X to O8'(Y), O5'(Y), O2'(Y) of molecule Y, respectively, and similar hydrogen bonds are observed between C2(Phe2), C2(Phe5), C2(Phe8) of molecule Y to O8'(X), O5'(X), O2'(X) of molecule X, respectively (Table I). The converse numbers associated with the atoms (donors and acceptors) involved in the hydrogen bonds represent the amazing regularity maintained to achieve the state of maximum possible hydrogen bonding (or weak interaction) between the two anti-parallel ambidextrous helices. Further, Phe with its planar aromatic side chain stacks against another Phe from the adjacent shape-complement helix leading to two `extended phenyl embrace' arrangements at the helix–helix interior (Figure 3). It is interesting that the average of equivalent temperature factors of the six atoms of phenyl rings of the Phe residues at the interface of the two antiparallel helices, and Y are lower than those of the Phe residues not involved in the interface. These values further support our observation that the networks of weak interactions observed between the two helices are strong enough to bring molecular association.
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Circular dichroism studies
CD spectroscopy has been used earlier to establish the screw sense of peptides containing dehydrophenylalanine residues (Pieroni et al., 1996). For the present decapeptide, CD studies were carried out in various solvents at room temperature. The CD spectra display couplets of bands which appear as a typical exciton splitting of the electronic transition of Phe chromophore at 280 nm. This type of splitting pattern originates from a rigid mutual disposition of two or more Phe residues, generally involved in a system of consecutive ß-turns, i.e. 310-helix (Pieroni et al., 1993). The most striking feature of the CD spectra of the decapeptide is the sign of the couplet. A negative CD couplet (– +), typical of a right-handed 310-helix with a negative band at 285 nm, a positive band at 260 nm and a crossover point at 275 nm, is observed for the decapeptide in dichloromethane, hexafluoro-2-propanol and chloroform (Figures 4 and 5). However, a positive CD couplet (+ –) with bands of opposite signs at 285 and 260 nm, characteristic of the left-handed screw sense of the helix (Pieroni et al., 1991), is also observed for the peptide in dimethylformamide and 80% methanol (Figures 4 and 5). The CD spectra of the decapeptide in chloroform with increasing concentration of methanol are indicative of the equilibrium shifting towards the left-handed conformer. Addition of methanol gradually appears to destabilize the right-handed form of the 310-helix. At a concentration of 60:40 (chloroform: methanol), almost equal proportions of the two conformers are present and, on further increase in methanol concentration, the left-handed helical conformation stabilizes in solution. Low molar ellipticity of the decapeptide in acetonitrile and trifluoroethanol may be attributed to the presence of both left-and right-handed conformers, simultaneously in solution, supporting the structure observed in the solid state. The equilibrium exhibited by the decapeptide may therefore shift towards the right-or left-handed conformer depending on the solvent conditions. Similar effects of solvent dependence on the handedness of the helix have also been reported by other workers (Pieroni et al., 1991).
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A CD band is observed at 320 nm that also changes sign with change in solvent. One reason for this band at 320 nm may be the weak electronic transition polarized along the short axis of the benzene ring. This contribution to the CD spectrum suggests that the benzene rings are not free to rotate, as expected, owing to the presence of the side chain C=C double bond. Its strong intensity also indicates that the phenyl rings are restricted to a preferred chiral disposition. However, there may be another explanation for the presence of the CD band at 320 nm. It is known that the dipole–dipole interaction between the transition moments polarized along the long axis of the chromophores leads to the CD couplet characterized by the positive band at 260 nm and the negative band at 290 nm, for a right-handed helix. Similarly, the dipole–dipole interaction between transition moments polarized along the short axis may lead to a second couplet with a negative band at 290 nm, overlapping with the previous negative band and a positive band at 320 nm. It may also be that both of these factors contribute to the presence of the band at 320 nm.
Solid-state and solution studies suggest that the energy barrier between the left-and right-handed conformers being less allows facile interconversion in solution and consequent association of the two conformers. Further, it appears that the 310-helical conformations and the shape complementarity of the conformers (X and Y) are the two key factors responsible for the stabilization of the C–H···O hydrogen-bonded Phe zipper structure. In 310-helical peptides (ideal), the side chains stack one over the other along the helical axis creating a column of protuberant side chains at 120° to each other, hence forming a groove between them. This makes the space for side chains from adjacent helices to interdigitate in the groove. In the present case the planar Phe side chains (hydrogen atoms in the same plane) with their favored disposition with respect to backbone are able to insert into the groove of the adjacent shape complement helix. Further, these interdigitated side-chains from shape complement helices are able to form good C–H···O hydrogen bonds with the backbone carbonyl oxygens of the adjacent helix. This provides a good example of shape assisted cooperative recognition of helices through weak interactions. This type of interdigitation may be described as a `wedge into a groove' arrangement as opposed to a `knobs into holes' arrangement (Crick, 1953) as proposed for -helices forming coiled coils.
Conclusion
The structure reveals an unusual association of ambidextrous 310-helices through strong C–H···O interactions. According to the `core hypothesis', the efficient packing of hydrophobic side chains in the interior primarily determines the native folded structure of globular proteins (Sander, 1994). Nature constructs this core mainly with the available hydrophobic residues from its toolbox of 20 amino acids. The hidden and plastic interactions involving hydrophobic groups play a substantial role in protein stability. In addition, the ability of a phenylalanine residue to lend extra stability to protein structures was pointed out by Burley and Petsko (Burly and Petsko, 1988). Moreover, almost all the de novo designed super secondary motifs containing only natural residues which fold/unfold cooperatively in water revealed the fact that aromatic residues play a substantial role in their stability (Baltzer, 1999; De Grado et al., 1999). In a recent study, Ramagopal et al. showed that aromatic interactions can be cleverly exploited in the design of helical hairpin motif, without polar/apolar patterning or disulfide bridges (Ramagopal et al., 2001). Similarly, it was shown that tryptophan zippers are capable of stabilizing small (12 residue) monomeric ß-hairpins (Cochran et al., 2001). All these results highlight the importance of weak interactions in molecular recognition and in the de novo design of mini-proteins, where a smaller number of buried hydrophobic residues are expected to play a major role.
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Notes |
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5 To whom correspondence should be addressed. E-mail: virander{at}icgeb.res.in
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Acknowledgments |
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The authors thank the Department of Science and Technology, India, for financial support and the Department of Biotechnology, India, for access to computers at the Bioinformatics and Interactive Graphic Facility. We thank Professor J.Shashidhara Prasad for providing access to the DST-supported National Single Crystal Diffractometer Facility at the University of Mysore. Ms P.Mathur thanks the CSIR, India, for a fellowship.
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Received September 11, 2001; revised January 10, 2002; accepted January 28, 2002.
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