Disulfide Bond and Topological Isomerization of the Conopeptide PNID: Disulfide Bonds with a Twist.

Abstract

Despite decades of research, efficient and accurate peptide and protein folding remains to be a problematic feat. This is largely due to difficulty in determining ubiquitous folding characteristics among diverse peptide sequences and in establishing an overarching theme in the folding process. Thus it is critical to the advancement of knowledge on the topic that novel sequences and frameworks of peptides continue to be studied. Conotoxins have proven to be excellent subjects of study as they are small, bioactive, and their activity is often highly dependent on achieving an appropriate fold. 􀁆/􀁏-Conotoxins have garnered significant interest due to their selective and inhibitory properties at the norepinephrine transporter (NET) (1,2). Here we present a novel 􀁆/􀁏-conotoxin, PnID, which unlike the previously reported members of this class, is expressed as two native disulfide isomers within the venom duct extract of Conus pennaceus. Additionally, synthetic production of this peptide resulted in one ribbon isomer and two distinct topological globular isomers (named PnID Isomer B and PnID Isomer RevB), in which their formation was later found to be individually controlled by the sequential order in which their two disulfide bonds were selectively formed. Only one of the globular isomers, PnID B, demonstrated a small amount activity at the monoamine transporters. The ribbon isomer, PnID A showed inhibition of the NET, with and IC50 of 10 μM. A second globular isomer (with reversed topology) PnID RevB, was separately produced and demonstrated no comparative inhibition. NMR analysis and structure calculations revealed that the occurrence of these novel topological isomers maybe due to a difference in two properties: (1) disulfide bond orientation, with retention of same connectivity and (2) backbone flexibility. A critical turn between residues 5-8 within PnID appears to be largely affected by disulfide bond formation, where the more active topology, PnID Isomer B possesses an increased chance of forming a 􀁊-turn, similar to the turn seen in 􀁆-conotoxin MrIA, whereas the inactive isomer, PnID Isomer RevB, appears to have a higher tendency to form an α-turn. The implication that disulfide bond connectivity alone may not be enough information for the efficient synthetic reproduction of all Conus derived toxins is novel and wide impacting; this then requires closer scrutiny to the physical orientation of disulfide bonds in respect to each other. These findings implicate how pharmacological selectivity and/or specificity may be influenced by the synthetic strategy in sequential selective disulfide bond formation. We believe these findings could have implications on the future of synthetically produced and rationally designed peptide drugs to enhance receptor-targeting selectivity and/or specificity.