Semienzymatic cyclization of disulfide-rich peptides using Sortase A

Abstract

Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.

Keywords: Cyclic Peptides; Cyclotides; Disulfide; Enzymatic Cyclization; Kalata B1; NMR; Peptide Chemical Synthesis; Peptides; Sortase A; Toxins.

FIGURE 1.

A, representation of the sequence and structure (PDB code 1JBL) of native SFTI-1; B, native kB1 (PDB code 1NB1), which is the prototypic cyclotide; C, native linear Vc1.1 (PDB code 2H8S), which has been cyclized by a 6-amino acid linker using native chemical ligation (3, 32). The disulfide bonding network is shown as sticks, with the cysteine residues numbered. Amino acids are represented by their one-letter code. D, the linear oxidized [GGG]kB1[TGG] contains a SrtA recognition sequence (LPVTG) and undergoes an intramolecular transpeptidation reaction catalyzed by SrtA to form cyclo-[GGG]kB1[T].

FIGURE 2.

A, RP-HPLC profile of [GGG]kB1[TGG] oxidation at 0 and 20 h. A major peak of linear precursor peptide and linear oxidized product is marked with a schematic representation of the peptide. B, time course analysis of the SrtA reaction progress monitored by RP-HPLC. Aliquots were taken from the reaction mixture at 0, 4, 8, 12, and 24 h and loaded onto an analytical RP-HPLC. C, MALDI-TOF analysis of linear precursor, linear oxidized [GGG]kB1[TGG], native kB1 peptide, and cyclic oxidized [GGG]kB1[T] (clockwise). Cyclo-[GGG]kB1[T] revealed a mass corresponding to the linear oxidized [GGG]kB1[TGG] minus two glycines and a water molecule, indicative of the formation of an amide bond between the N and C termini. D, analytical RP-HPLC profile of purified cyclo-[GGG]kB1[T] (top) and co-injection of cyclo-[GGG]kB1[T] and native kB1 (bottom) to demonstrate their only slightly different retention times.

FIGURE 3.

Hα secondary shift comparison of native kB1 (empty squares with dashed line) and cyclo-[GGG]kB1[T] (filled squares with solid line). The Hα secondary shifts were calculated by subtracting random coil shifts (81) from the experimental Hα shifts. The box highlights loop 6, where the extra residues TGGG were introduced by the SrtA-mediated cyclization.

FIGURE 4.

A, superimposition of the 20 lowest energy structures of cyclo-[GGG]kB1[T] shown in blue with disulfide bonds shown in orange. B, comparison of cyclo-[GGG]kB1[T] in blue with native kB1 in red (PDB code 1NB1) by superimposition of residues 1–24 of cyclo-[GGG]kB1[T] against residues 1–24 of the native kB1 structure. Disulfide bonds are shown in orange, highlighting the similarities between the two structures across loop 1–5 and the increased flexibility of loop 6 by insertion of the SrtA sorting motif. Root mean square deviation across heavy backbone atoms for residues 1–24 of cyclo-[GGG]kB1[T]/1–24 of kB1 is 0.461.

FIGURE 5.

Hemolytic and serum stability assay on cyclo-[GGG]kB1[T]. A, percentage of hemolysis by cyclo-[GGG]kB1[T] compared with native kB1 included as a positive control. B, percentage of peptide remaining after 0-, 1-, 2-, 3-, 5-, 8-, 11-, and 24-h incubation in human serum. The data are presented as means ± S.D.

FIGURE 6.

A, cyclization strategy for [G]Vc1.1[GLPETGGS] and [GG]SFTI-1[LPETGG] by SrtA. B, retention time and MALDI-TOF analysis of linear precursor [G]Vc1.1[GLPETGGS], cyclic reduced, and cyclic oxidized [G]Vc1.1[LPET]. The peptide status is shown with schematic representations. C, one-pot reaction of oxidation and cyclization of [GG]SFTI-1[LPETGG]. Shown is a time course analysis of the SrtA reaction by RP-HPLC (left) and MALDI-TOF data (right) of linear precursor [GG]SFTI-1[LPETGG] and cyclo-[GG]SFTI-1[LPET]. D, Hα secondary shift comparison of cyclo-[GG]SFTI-1[LPET] and wild type SFTI-1. E, Hα secondary shift comparison of cyclo-[G]Vc1.1[GLPET], wild type Vc1.1, and the orally active analgesic peptide cVc1.1 (3). The sorting motif is shown in a dashed box.