Topological isomers of a potent wound healing peptide: Structural insights and implications for bioactivity

There are numerous examples of topological isomers in organic chemistry, but such isomers are rare in disulfide-rich peptides. Here, we characterize two structurally well-defined topological isomers in a peptide (GRN-P4A) containing the mini-granulin fold. The mini-granulin fold is emerging as an important disulfide-rich structural motif with promising implications for the enhancement of wound-healing strategies. The two topological isomers of GRN-P4A have well-defined structures that do not interconvert, and although they have the same disulfide bond connectivity and similar overall structures, they have structural differences related to the first inter-cysteine loop. These structural changes influence the bioactivity as the isomers have significant differences in their cell proliferation activity. Prediction of the structure using AlphaFold3 identified the correct disulfide bond connectivity, but the structure of Loop 1 was similar to the less abundant isomer of GRN-P4A and did not indicate topological isomerization. These topological isomers introduce significant complexity to the understanding of folding mechanisms in this class of peptides, and potentially other disulfide-rich peptides, offering valuable insights for protein design and engineering by presenting a novel topological fold-switching mechanism. Additionally, they hold practical implications to produce GRN-P4A, given its promising potential as a wound-healing agent.