The structural integrity of human TFF1 under reducing ...

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Highlights

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  Human trefoil factor 1 (TTF1) protein is stabilized by three intramolecular disulfide bonds.

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  Complete reduction of disulfide bonds of TFF1 occurs only in large excess of reducing agent.

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  TFF1 shows enhanced stability compared to reference peptides BPTI and linaclotide.

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  MD simulations reveal details of non-covalent stabilization across different redox states.

Abstract

The trefoil factor family (TFF) comprises three secretory peptides (TFF1, TFF2, TFF3) that regulate diverse physiological processes to maintain gastrointestinal mucosal integrity and homeostasis. The TFF domain is stabilized by six conserved cysteine residues forming three intramolecular disulfide bonds. In this work, we investigated human TFF1 domain stability against increasing concentrations of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Experiments revealed high resistance of the disulfide bonds within the TFF1 domain to reduction compared to two reference peptides with similar three-disulfide frameworks, namely the bovine pancreatic trypsin inhibitor (BPTI) and the peptide drug linaclotide. Full reduction of TFF1 was only achieved with a large excess of TCEP (150-fold), and no partially reduced intermediates were observed, supporting a compact TFF1 domain. This TFF1 domain stability was supported by extensive all-atom molecular dynamics simulations for a total of 24 μs of all possible combinatorial states of disulfide bond reduction. Despite minor structural and conformational changes observed upon reduction, the domain substantially retained its overall compactness and solvent exposure when only one or two disulfide bonds were removed. The reduced cysteine residues did not undergo large structural rearrangements and remained buried. The loss of covalent disulfide bonds upon reduction was counterbalanced through persistent non-covalent interactions. These molecular simulations explained why TFF1 could not be partially reduced and alkylated during the experiments despite titrating different TCEP concentrations in the presence of alkylating agents. Our findings provide the first insights into the remarkable stability of the human TFF domain under reducing conditions, supporting its functional resilience upon expression and secretion throughout the human body.

Keywords

Trefoil factor family

Disulfide bond stability

Reductive stability

Molecular dynamics simulation

Data availability

Data will be made available on request.

1

Both authors contributed equally.

© 2025 The Authors. Published by Elsevier B.V.