Poster Presentation The 16th Australian Peptide Conference 2025

Dissecting the trefoil domain folding process through directed disulfide bond formation (#108)

John Raju 1 , Gene Hopping 1 , Markus Muttenthaler 1 2
  1. The University of Queensland, Australia, Brisbane, QUEENSLAND, Australia
  2. Institute of Biological Chemistry, University of Vienna, Vienna, Austria

The Trefoil Factor Family (TFF) comprises three endogenously expressed gut peptides (TFF1/2/3) that are recognised for preserving gastrointestinal integrity through epithelial repair, mucosal maintenance, and tissue homeostasis. These protective functions hold therapeutic promise for gastrointestinal disorders such as inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), and celiac disease, which are characterised by epithelial injury, inflammation, and mucosal disruption.1, 2 All three TFF members (TFFs) have been investigated for their biological activity using gene knockout techniques, yet reliable synthetic access to the individual TFFs was only recently achieved,3, 4 enabling more comprehensive structure-activity relationship studies.

The TFFs’ bioactivity and high stability depend on their 3D structure, which is stabilised by three disulfide bonds, giving rise to a highly compact three-loop trefoil domain fold.5 The trefoil domain includes two vicinal cysteines, and their connectivity decides between the described trefoil domain fold or the evolutionary common inhibitory cystine knot (ICK) fold, and it is unclear how such a different connectivity would affect folding, 3D structure, stability and bioactivity.

In this study, we use directed disulfide bond folding strategies coupled to analytical HPLC, proteomics, CD and NMR techniques to investigate this vicinal cysteine region that can give rise to native and non-native folding of TFF3. Understanding the folding process and the impact of cysteine connectivity is important for the chemical synthesis, correct folding and analysis of TFF analogues, particularly when non-native amino acids are being introduced for probe and drug development. Additionally, this study will provide fundamental insights into the direct comparison of two very important folds, namely the trefoil vs the ICK fold.

 

  1. Emidio, N. B.; Brierley, S. M.; Schroeder, C. I.; Muttenthaler, M. Structure, Function, and Therapeutic Potential of the Trefoil Factor Family in the Gastrointestinal Tract. ACS Pharmacology & Translational Science 2020, 3 (4). DOI: 10.1021/acsptsci.0c00023.
  2. Emidio, N. B.; Hoffmann, W.; Brierley, S. M.; Muttenthaler, M. Trefoil Factor Family: Unresolved Questions and Clinical Perspectives. Trends in Biochemical Sciences 2019, 44 (5), 387-390. DOI: 10.1016/j.tibs.2019.01.004.
  3. Braga Emidio, N.; Baik, H.; Lee, D.; Sturmer, R.; Heuer, J.; Elliott, A. G.; Blaskovich, M. A. T.; Haupenthal, K.; Tegtmeyer, N.; Hoffmann, W.; et al. Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action. Chem. Commun. 2020, 56, 6420-6423. DOI: 10.1039/d0cc02321c.
  4. Braga Emidio, N.; Meli, R.; Tran, H. N. T.; Baik, H.; Morisset-Lopez, S.; Elliott, A. G.; Blaskovich, M. A. T.; Spiller, S.; Beck-Sickinger, A. G.; Schroeder, C. I.; Muttenthaler, M. Chemical Synthesis of TFF3 Reveals Novel Mechanistic Insights and a Gut-Stable Metabolite. Journal of Medicinal Chemistry 2021, 64 (13), 9484-9495. DOI: 10.1021/acs.jmedchem.1c00767.
  5. Thim, L. A new family of growth factor-like peptides. 'Trefoil' disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins). FEBS Lett 1989, 250 (1), 85-90. DOI: 10.1016/0014-5793(89)80690-8.