Solution Structure Studies 
of
Trefoil Factor Family Proteins

Department of Biosciences, University of Kent
&
Molecular Structure Division, National Institute for Medical Research

The Trefoil Factor Family

The 'trefoil' sequence motif is a characteristic sequence pattern of about 40 residues - containing six cysteines with well conserved spacing - which has been found, either singly or as a repeat, in about a dozen extracellular proteins. A sub-class of these proteins that consist predominantly of trefoil domains are referred to as trefoil proteins. Studies of the three dimensional structure of a few of these trefoil proteins have been completed in the past few years. These studies show that the conserved cysteines in each domain form three disulphide bonds with  1-5, 2-4, 3-6 pairings, producing a compact three loop structure that has only a small amount of secondary structure (a short helix packed against a two strand anti-parallel beta sheet). The topology of the fold is presently thought to be unique to this particular sequence motif.

Three mammalian trefoil proteins have been identified; spasmolytic polypeptide (TFF2) contains two domains and intestinal trefoil factor (TFF3) and pS2 (TFF1) both contain a single domain. They are predominantly secreted by mucus cells of the gut, where they are known to be actively involved in protecting against ongoing damage by agents such as non-steroidal anti-inflammatory drugs, and are also involved in repair of the ulcerative damage. Additionally both TFF1 and TFF3 have been found to be over expressed in several cancer cell lines.  Their molecular targets are, as yet, unknown.

A list of currently known trefoil domains is maintained at this site, and the trefoil peptide homepage is maintained by Andrew Giraud at the University of Melbourne.
 

Structure of human TFF1

TFF1 (aka pS2 or pNR-2 or BCEI protein) was the first trefoil protein to be discovered. It was discovered in 1984 by virtue of its estrogen regulation in human breast cancer cells. It is secreted as a 60 residue protein that contains a single trefoil motif. Outside the region of the trefoil motif it has a seventh cysteine residue near the C-terminus that is able to link two TFF1 molecules to form a disulphide bonded dimer.

In collaboration with Felicity May and Bruce Westley at the University of Newcastle (who made the protein), the group at NIMR, principally Vladimir Polshakov and Mark Williams, recently determined a high resolution structure of a mutant TFF1 - in which the seventh Cys was mutated to Ser to prevent dimerisation. A summary of the structure of this monomeric form and a recent poster with more detail are available. Mark Williams is currently completing the structure determination of the disulphide linked dimeric form of the protein.
 

Structure of porcine TFF2

TFF2 (aka spasmolytic polypeptide) was the second trefoil protein to be discovered, originally as a contaminant in porcine insulin. It secreted as a 106 residue protein containing two trefoil sequence motifs.

The solution structure of TFF2 was the first solution structure of a trefoil factor to be determined, being  solved at the NIMR, principally by Mark Carr, in 1993. It is consists of two trefoil domains held in close proximity by an additional intra-molecular disulphide bond.  A summary of the structure is available.
 

Structure of human TFF3

TFF3 (aka intestinal trefoil factor) is a another 60 residue secreted protein which contains a single trefoil motif and is able to form disulphide linked dimers through a C-terminal cysteine. However, the C terminal extension is much shorter than than TFF1 and the amino acid substitution pattern suggests that it probably has rather different binding properties.

Mark Carr now leads an independent research group at the University of Kent, which has recently determined the structure of the monomeric form of TFF3, principally through the efforts of Xavier Lemercin (based at the NIBSC) and Fred Muskett (now at NIMR). The proteins for the pTFF2 and hTFF3 studies were supplied by Lars Thim, who's group at Novo Nordisk, Copenhagen, is involved in x-ray crystallographic studies of the trefoil proteins.
 

Active Researchers

Dr Mark D. Carr                   - Lecturer in Structural Biology, Dept. of Biosciences, University of Kent, Canterbury
                                                - All trefoil factors - particularly, porcine TFF2 and human TFF3

Dr Jim Feeney                    - Head of the Molecular Structure Division, National Institute for Medical Research
                                                - All trefoil factors - particularly, human TFF1 and TFF2

Dr Xavier Lemercin            - Staff Scientist, Laboratory of Molecular Structure, National Institute for Biological Standards and Control
                                                 - human TFF3

Dr Fred W. Muskett            - PostDoc, MRC Biomedical NMR Centre, NIMR
                                                 - human TFF3

Dr Vladimir I. Polshakov    - Visiting Worker at NIMR and Howard Hughes International Scholar at the CPRI, Moscow
                                                 - human TFF1

Dr Mark A. Williams           - Visiting Worker at NIMR and Senior Research Fellow at Dept. of Biochemistry & Molecular Biology, University College, London
                                                 - human TFF1 and trefoil factor familial traits
 

Publications

Carr, M.D. (1992). H-1 NMR based determination of the secondary structure of porcine pancreatic spasmolytic polypeptide - one of a new family of trefoil motif containing cell-growth factors. Biochemistry 31, 1998-2000.

De, A., Brown, D.G., Gorman, M.A., Carr, M., Sanderson, M.R. and Freemont, P.S. (1994). Crystal-structure of  a disulphide linked treoil motif found in a large family of putative growth-factors. PNAS 91, 1084-1088.

Carr, M.D., Bauer, C.J., Gradwell, M.J., Feeney, J. (1994). Solution structure of a trefoil-motif-containing cell-growth factor, porcine spasmolytic protein. PNAS 91, 2206-2210.

Polshakov, V.I., Frenkiel, T.A., Westley, B., Chadwick, M., May, F., Carr, M.D. and Feeney, J. (1995). NMR-based structural studies of the pNR-2/pS2 single domain trefoil peptide: Similarities to porcine spasmolytic peptide and evidence for a monomeric structure. European Journal of Biochemistry233, 847-855.

Polshakov, V. I., Williams, M. A., Gargaro, A. R., Frenkiel, T. A., Westley, B. R., Chadwick, M. P., May, F. E. B. and Feeney, J. (1997). High-resolution solution structure of human pNR-2/pS2: a single trefoil motif protein. Journal of Molecular Biology 267, 418-432.
 

Contact Information

General inquiries (re: collaboration, materials etc.) should be addressed to the Group Leader (Mark Carr or Jim Feeney) who seems most appropriate. Specific questions about the solved structures may be better addressed to the people who solved them.