Mutagenesis of bracelet cyclotide hyen D reveals functionally and structurally critical residues for membrane binding and cytotoxicity

Abstract

Cyclotides have a wide range of bioactivities relevant for agricultural and pharmaceutical applications. This large family of naturally occurring macrocyclic peptides is divided into three subfamilies, with the bracelet subfamily being the largest and comprising the most potent cyclotides reported to date. However, attempts to harness the natural bioactivities of bracelet cyclotides and engineer-optimized analogs have been hindered by a lack of understanding of the structural and functional role of their constituent residues, which has been challenging because bracelet cyclotides are difficult to produce synthetically. We recently established a facile strategy to make the I11L mutant of cyclotide hyen D that is as active as the parent peptide, enabling the subsequent production of a series of variants. In the current study, we report an alanine mutagenesis structure-activity study of [I11L] hyen D to probe the role of individual residues on peptide folding using analytical chromatography, on molecular function using surface plasmon resonance, and on therapeutic potential using cytotoxicity assays. We found that Glu-6 and Thr-15 are critical for maintaining the structure of bracelet cyclotides and that hydrophobic residues in loops 2 and 3 are essential for membrane binding and cytotoxic activity, findings that are distinct from the structural and functional characteristics determined for other cyclotide subfamilies. In conclusion, this is the first report of a mutagenesis scan conducted on a bracelet cyclotide, offering insights into their function and supporting future efforts to engineer bracelet cyclotides for biotechnological applications.

Keywords: NMR; cancer cell cytotoxicity; cyclic peptide; cyclotide; peptide chemical synthesis; peptide-membrane interaction; site-directed mutagenesis; surface plasmon resonance.

Figure 1

Three-dimensional structures of prototypical cyclotides of the two major subfamilies and an illustration of cis- and trans-proline conformations. Three-dimensional structures of the prototypical cyclotides from Möbius (A) and bracelet (B) cyclotide subfamilies. The NMR solution structures of kalata B1 and cycloviolacin O2 are from PDB files 1NB1 and 2KNM, respectively (12, 13). C, alignment of exemplary cyclotides from bracelet and Möbius subfamilies. The orange and black lines, respectively, represent the disulfide bonds and the cyclic backbone. Cysteine residues are highlighted in yellow. The Pro residue in loop 5 of Möbius cyclotides that adopts a cis conformation is in red. The structural features of the prototypical cyclotides kB1 and cyO2 are highlighted in purple or blue. Each β-strand is depicted by an arrow, and the 3₁₀ helix is depicted by a short helix (blue).

Figure 2

Structural characterization of hyen D and I11L using NMR.A, superimposition of the 20 lowest-energy models of the hyen D solution structure computed from NMR data. B, schematic view of the secondary structure of hyen D. Hydrogen bonds are shown as gray-dashed arrows, β-strands are shown as black arrows, and the 3₁₀ helix is shown as a black ribbon. C, structure comparison between cycloviolacin O1 (hot pink), cyO2 (cyan), kalata B5 (pink), and hyen D (green). D, secondary Hα chemical shifts of I11L (orange) compared to hyen D (green). Residue number, loop position, and Roman numeral for each cysteine are provided above the sequence. Secondary Hα chemical shifts provide information on secondary structure as indicated by Wishart et al (29). A group of four or more consecutive residues with secondary Hα chemical shifts less than −0.1 is likely to adopt a helical structure, whereas a cluster of three or more residues with values more than 0.1 is likely to be a β-strand.

Figure 3

Hα secondary NMR chemical shifts of I11L and its Ala mutants compared to I11L and native hyen D. The Hα chemical shifts for Phe-14 and Thr-15 are missing in the spectra for most peptides. Cysteine residues are highlighted in yellow.

Figure 4

Sequence logo of functionally critical residues within typical bracelet cyclotides. For better alignment, the sequence logo of loop 3 was separated into six- and seven-residue subgroups. The conserved structurally critical residues are highlighted in orange, while hydrophobic residues are highlighted in dark green.

Figure 5

Schematic representation of the membrane-binding orientation of hyen D and kB1. The hydrophobic face and bioactive face are surrounded by solid and dashed lines, respectively. The bioactive faces of both molecules are highlighted in pink. A, the hydrophobic face for hyen D is shown in green. Residues in light green assist in membrane interaction, while residues in dark green are deeply inserted into the membrane. B, the hydrophobic face for kB1 is shown in purple. The faint blue area represents the potential location of the cell membrane depicting the interaction of the hydrophobic face of hyen D (or kB1) with the membrane.