Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide
© Acharya et al; licensee BioMed Central Ltd. 2007
Received: 10 November 2006
Accepted: 01 August 2007
Published: 01 August 2007
The de novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success of these studies relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α, β-dehydroamino acids, especially α, β-dehydrophenylalanine (ΔPhe) comes in use for spawning well-defined structural motifs. Introduction of ΔPhe induces β-bends in small and 310-helices in longer peptide sequences.
The present report is an investigation of the effect of incorporating two glycines in the middle of a ΔPhe containing undecapeptide. A de novo designed undecapeptide, Ac-Gly1-Ala2-ΔPhe3-Leu4-Gly5-ΔPhe6-Leu7-Gly8-ΔPhe9-Ala10-Gly11-NH2, was synthesized and characterized using X-ray diffraction and Circular Dichroism spectroscopic methods. Crystallographic studies suggest that, despite the presence of L-amino acid (L-Ala and L-Leu) residues in the middle of the sequence, the peptide adopts a 310-helical conformation of ambidextrous screw sense, one of them a left-handed (A) and the other a right-handed (B) 310-helix with A and B being antiparallel to each other. However, CD studies reveal that the undecapeptide exclusively adopts a right-handed 310-helical conformation. In the crystal packing, three different interhelical interfaces, Leu-Leu, Gly-Gly and ΔPhe-ΔPhe are observed between the helices A and B. A network of C-H...O hydrogen bonds are observed at ΔPhe-ΔPhe and Gly-Gly interhelical interfaces. An important feature observed is the occurrence of glycine zipper motif at Gly-Gly interface. At this interface, the geometric pattern of interhelical interactions seems to resemble those observed between helices in transmembrane (TM) proteins.
The present design strategy can thus be exploited in future work on de novo design of helical bundles of higher order and compaction utilizing ΔPhe residues along with GXXG motif.
De novo protein design endeavors to construct novel polypeptide sequences that fold into well-defined secondary and tertiary structures resembling those found in native proteins. Many de novo design strategies have relied on the known penchants of protein amino acids to espouse various secondary structures leading to several remarkable achievements [1–4]. Alternatively, the amalgamation of conformationally restricted, non-protein amino acids by chemical synthesis has led to triumphant designs of secondary and super secondary structures that mimic proteins [5, 6]. In this regard, the ability of α, β-dehydrophenylalanine (ΔPhe) to induce β-bends in small and 310-helices in longer peptide sequences has been well studied [7–18]. The presence of dehydroresidues in peptides confers altered bioactivity as well as increased resistance to enzymatic degradation . Recently designed super secondary structures such as ΔPhe zippers and helical hairpins highlight the potential of ΔPhe to introduce long-range interactions in peptides and it has been noted that the geometry of a 310-helix brings ΔPhe residues at i and i+3 position into a stacking arrangement and the structurally planar ΔPhe side-chains interdigitate to assist the cooperative recognition of helices [5, 17, 20]. In proteins, there is a wide interplay of weak non-covalent interactions between secondary structural elements, to achieve stability and overall compaction. In this context, in transmembrane proteins it is observed that glycine residues promotes close approach of helices, which permits not only favourable vander Waals interactions of surrounding side chains, but also in many cases, encourage interhelical Cα-H...O hydrogen bonds [21–25]. Interestingly, it has been found that the GXXXG motif elicits a level of self-association in putative transmembrane helices and the three-residue spacing between both glycines proves to be optimal for the interaction. In an attempt to mimic similar interactions and geometric features, we designed and synthesized an undecapeptide, Ac-Gly1-Ala2-ΔPhe3-Leu4-Gly5-ΔPhe6-Leu7-Gly8-ΔPhe9-Ala10-Gly11-NH2. Its structural features were characterized using X-ray diffraction and Circular Dichroism spectroscopy. ΔPhe residues and glycine residues as GXXG motif were at a two-residue spacer to give rise to a 310-helical conformation. Thus the peptide incorporates two GXXG like motif (Gly5-ΔPhe6-Leu7-Gly8 and Gly8-ΔPhe9 Ala10-Gly11) motif in the helix region and one GXXXG (Gly1-Ala2-ΔPhe3-Leu4-Gly5) motif near the N-terminus. The bulky leucine residues were placed in middle of the helical segment to ensure that the peptide folds into a right-handed screw sense. A 310-helical conformation of ambidextrous screw sense is established by X-ray diffraction. However, CD studies reveal that a right-handed 310-helical conformation dominate in solution. The preponderance of the right-handed 310-helical conformer is also confirmed using energy calculation studies [Additional file 1]. An unanticipated observation of ambidexterity of the peptide helices in the crystal structure demonstrates the influence of global interactions on the coexistence of left and right-handed helices in the crystal structure. This is a novel observation of a 310-helical dehydroundecapeptide mimicking interhelical interactions as seen amongst transmembrane helices.
Results and Discussion
Data collection and Refinement parameters for Ac-Gly-Ala-ΔPhe-Leu-Gly-ΔPhe-Leu-Gly-ΔPhe-Ala-Gly-NH2.
C55 H70 N12 O12·3H2O
(1091 + 54) Da
a = 11.2555(6) Å, b = 12.5450(6) Å, c = 21.6444(14) Å α = 75.460(2)°, β = 89.369(2)°,γ = 80.988(5)°
Z (molecules/unit cell)
1.241 g cm-3
(λ = 0.92015 Å)
7057 [|Fo| > 4σ(|Fo|)]
Full-matrix least-squares refinement on |Fo|2 using SHELXL97
Number of parameter refined
0.0667 (for [|Fo| > 4σ(|Fo|)])
0.1853 (for all unique reflections)
Residual electron density
Max. = 0.41 e/Å 3
Min = -0.31 e/Å 3
Torsion angles (°) for peptide.
Intrahelical hydrogen bonds observed in the crystal structure of Peptide Ac-Gly-Ala-ΔPhe-Leu-Gly-ΔPhe-Leu-Gly-ΔPhe-Ala-Gly-NH2.
Conformer A (left-handed 310-helix)
Conformer B (right-handed 310-helix)
Intermolecular hydrogen bonds observed in the crystal structure of the peptide.
x+1, y-1, z
x, y, z+1
x+1, y, z
x+1, y, z
x-1, y+1, z
x, y, z-1
x-1, y, z+1
x-1, y, z+1
x-1, y, z
x-1, y, z
Circular Dichroism studies
The present peptide, Ac-Gly-Ala-ΔPhe-Leu-Gly-ΔPhe-Leu-Gly-ΔPhe-Ala-Gly-NH2, provides the first example of stability and compaction in interacting helices when glycine residues are incorporated in the middle of the peptide sequence. The incorporation of glycines in the form of GXXG motif along with ΔPhe residue at two-residue spacer has helped in maintaining the 310-helical conformation in both solid as well as solution state. The amalgamation of GxxG motif has not only facilitated the helices to come close at the Gly-Gly interhelical interface but also promoted the formation of glyzine zipper, where a zipper like arrangement of Cα-H...O hydrogen bonds is observed. The occurrence of weak C-H...O hydrogen bonds at ΔPhe-ΔPhe interface along with occurrence of main chain to main chain Cα-H...O hydrogen bonds consistently at three residue intervals at Gly-Gly helical interface involving GXXG motifs seems to impart molecular association and stabilization to the interacting helices. The phenomenon of molecular association leading to stabilization of the 310-helical conformer is also confirmed by the solution state study. The present design can encourage the peptide designers in pursuing the ambitious goal of de novo design of helical bundles of higher order and compaction utilizing ΔPhe residues along with GXXG motifs.
The peptide crystals were grown by the slow evaporation of peptide solution (1:1 v/v) in ethanol and acetone mixture. The X-ray diffraction data was collected using a suitable crystal cryo cooled to 100 K in synchrotron radiation source, at beam line X9A, Brookhaven National Laboratory. The structure was solved by direct method using SHELXS and was refined using full matrix least square refinement employed in SHELXL . The hydrogen atoms were fixed using stereochemical criteria and were allowed to ride on parent atoms. The crystallographic data of the present peptide is deposited in CCDC (CCDC289231).
Circular Dichroism studies
CD spectra were recorded on a JASCO J-720 CD spectropolarimeter. The spectra were acquired between 220–330 nm (0.1 cm cell, peptide concentration ~100 μM) at 0.1 nm intervals with a time constant of 4 seconds and a scan speed of 200 nm/min and averaged over 6 separate scans. The spectra obtained were baseline corrected and smoothed. Peptide concentration was determined using the molar extinction coefficient of ΔPhe (~19,000 M-1cm-1). CHCl3-methanol titration was carried out. CD spectra were recorded at different concentrations of SDS and also at different percentage of TFE/water. The CD spectra were recorded in 40% TFE/water at variable temperatures.
The energy minimization for the present peptide was performed using the SYBYL software package (version 7.0) (1). The force field used was AMBER7 FF99 implemented in SYBYL. The convergence criterion of 0.05 kcal/mol (Å) as well as the non-bonded cut-off distance was set to 8Å. The partial charges on protein residues were AMBER7 F99 all-atom charges. A value of 1 was set out for dielectric constant for these peptides. The details of energy calculation values are given as additional file 2.
- Rinkamide MBHA resin:
Sodium dodecyl sulphate
Thin layer chromatography
The financial support from the Department of Science and Technology, India is acknowledged. The financial support from WHO, is also acknowledged. We would like to thank Prof. Faizan Ahmad at Jamia Millia Islamia for his consent in using the Circular Dichroism facility in his laboratory. We thank department of Biotechnology, India for access to facilities at Bioinformatics and interactive graphics facility, I.I.Sc, Bangalore. RA would like to thank IBM-CAS fellowship.
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