Chirality transmission in macromolecular domains

Abstract

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.

Fig. 1. Schematic of optical-tweezers set up for high-throughput single-molecule assay.

The inter-strand helical sense match/mismatch effects between the two macromolecular domains are shown in the bottom left inset in which the D-DNA triplex is marked in cyan rectangle, L-peptide coiled-coil in orange ellipse and D-peptide coiled-coil in purple ellipse. M and P stand for left-handed and right-handed inter-strand helical senses, respectively. The clash symbol depicts the helical sense mismatch state, which has unfavourable energy cost and thus is marked by a red-cross. The no clash symbol represents the helical sense match state, which should not influence the stability and thus is marked by a green check.

Fig. 2. POC synthesis.

A Conjugating two stereoisomeric azidopeptides to ON-BCNs via strain-promoted alkyne-azide cycloaddition to obtain L,D-POC1-18 and D,D-POC1-15. B Sequence of L-azidopeptide, D-azidopeptide and ONs. C Different linker lengths were employed to furnish the BCN function either on the 5′-end or on the 3′-end of ONs. Natural amino acids are denoted in upper cases while unnatural ones are in lower cases. Thus, Y, V, L, E, S, K, Q, A, G are L-amino acids whereas y, v, l, e, s, k, q, a, g are D-amino acids. A, G, C, and T are natural DNA monomers. Key: The two-letter prefix before POCs: The first letter indicates the chirality of the peptide while the second letter indicates the chirality of the ON.

Fig. 3. Effect of TFO or D,D-POC1 on the mechanical stability of the DNA duplex or the chimeric POC duplex D,D-POC(2 + 3).

Schematic diagrams, typical force-extension curves and corresponding force histograms of (A) DNA duplex control, (B) D,D-POC(2 + 3), (C) L,D-POC(2 + 3), (D) DNA triplex control, (E) D,D-POC(1 + 2 + 3). TFO = 3′-TTTTCCCTCTCTCT. Note: red and black traces in a force-extension curve represent stretching and relaxing events, respectively. N and n depict total numbers of features and distinct molecules, respectively.

Fig. 4. Comparative study of bound % for the L- and D-peptide with different linker lengths.

A Rupture force histograms for the L-peptide D-DNA (left) and D-peptide D-DNA (right) with varying linker length (in number of bonds). B Comparison of bound % for D-peptide D-DNA and L-peptide D-DNA. Data are presented as mean ± SD for n = 4 independent experiments. **P = 0.0085, ***P = 3.9 × 10⁻⁵ for the 21 bonds and P = 2.4 × 10⁻⁵ for the 24–25 bonds, ns: nonsignificant (two tailed unpaired t-test). C Percentage contribution of chirality clash and templated binding effects for the trimeric L-peptide D-DNA with varied linker length (see Methods for calculation). D Rupture force histograms for the L-peptide D-DNA (L,D-POC(2 + 3), 16 bonds) bound with a TFO conjugated with L-peptide (L,D-POC16, 44–45 bonds, left) or with D-peptide (D,D-POC1, 16 bonds, right). N and n depict total numbers of features and distinct molecules, respectively.

Fig. 5. MD simulation.

Packing of the linker region in the MD snapshots used for the SAXS prediction for D,D-POC(7 + 8 + 9)*(a–c) and for L,D-POC(7 + 8 + 9)* of our previous work (d–f). a, d Side views show dynamic stacking interaction between a linker triazole moiety (boxed grey in (a), boxed blue in (d)) and DNA triplex terminal bases. Terminal tyrosines are shown in stick representation. DNA atoms are shown in ball and stick representation. b, e Isolated views of the linker/base stacking interactions where the linker is shown in stick representation. c, f Top views along coiled-coil axis showing packing of the linkers and tyrosine side chains. Except for the tyrosines, coiled-coil atoms are not shown but collectively represented by the C-alpha atoms of the terminal tyrosines (red, blue, grey spheres with black outlines). White arrows indicated the tyrosine side chain which transiently separated one linker strand (grey) from the other two hydrophobically packed linker strands (blue and red) in the chosen snapshot.

Fig. 6. The stability of POC triple helices is influenced by the helical sense mismatch/match and the templated binding effect.

D-DNA triplex is marked in cyan rectangle, L-peptide coiled-coil in orange ellipse and D-peptide coiled-coil in purple ellipse. M and P stand for left-handed and right-handed inter-strand helical senses, respectively. (Top: The clash symbol means the helical sense mismatch state, which may result in unfavourable energy cost and thus is marked in a red-cross. The no clash symbol represents the helical sense match state, which should not influence the stability and thus is marked in a green check).