Self-Assembly of Catenanes from Lasso Peptides

Abstract

Lasso peptides exist naturally in a threaded state as [1]rotaxanes, and we reasoned that lasso peptides cleaved in their loop region could serve as building blocks for catenanes. Mutagenesis of the lasso peptide microcin J25 (MccJ25) with two cysteine residues followed by cleavage of the peptide with trypsin led to a [2]rotaxane structure that self-assembled into a [3]catenane and [4]catenanes at room temperature in aqueous solution. The [3]catenane represents the smallest ring size of a catenane composed solely of polypeptide segments. The NMR structure of the [3]catenane was determined, suggesting that burial of hydrophobic residues may be a driving force for assembly of the catenane structure.

Figure 1

Structure and cleavage of MccJ25. A) Schematic of wild-type MccJ25. B) Sequence of MccJ25 RCC with amino acid substitutions highlighted in red. When subjected to trypsin cleavage, the [2]rotaxane (right) is formed.

Figure 2

Assembly of [3] and [4]catenanes from MccJ25 RCC. A) HPLC traces showing reaction of lasso peptide (top) with trypsin after 25 hours (middle) and after 50 hours (bottom). B) Mass spectra of the three major peaks from 50 hour reaction, where each peak was individually purified. Top is for the peak at retention time 14.67 minutes, middle is for the peak at 14.83 minutes, and bottom is for the peak at 15.17 minute retention time. C) Schematic of the [3] and [4]catenanes.

Figure 3

NMR structure of the [3]catenane. A) Lowest energy structure of the [3]catenane showing the peptide backbone with the original MccJ25 rings in purple, and the newly formed central disulfide bonded ring in turquoise. B) Rotation of figure A by approximately 90 degrees. C) The 20 lowest energy structures, where the Ile-17 and Phe-19 residues from both monomers are shown as lines, highlighting the inter-monomer interactions identified in our NOEs.