HIGH-RESOLUTION SOLUTION STRUCTURE OF HUMAN PNR-2 PS2 - A SINGLE TREFOIL MOTIF PROTEIN/

pNR-2/pS2 is a 60 residue extracellular protein, which was originally discovered in human breast cancer cells, and subsequently found in oth er tumours and normal gastric epithelial cells. We have determined the three-dimensional solution structure of a C58S mutant of human pNR-2/ pS2 using 639 distance and 137 torsion angle constraints obtained from analysis of multidimensional NMR spectra. A series of simulated annea ling calculations resulted in the unambiguous determination of the pro tein's disulphide bonding pattern and produced a family of 19 structur es consistent with the constraints. The peptide contains a single ''tr efoil'' sequence motif, a region of about 40 residues with a character istic sequence pattern, which has been found, either singly or as a re peat, in about a dozen extracellular proteins. The trefoil domain cont ains three disulphide bonds, whose 1-5, 2-4 and 3-6 cysteine pairings form the structure into three closely packed loops with only a small a mount of secondary structure, which consists of a short alpha-helix pa cked against a two-stranded antiparallel beta-sheet. The structure of the domain is very similar to those of the two trefoil domains that oc cur in porcine spasmolytic polypeptide (PSP), the only member of the t refoil family whose three-dimensional structure has been previously de termined. Outside the trefoil domain, which forms the compact ''head'' of the molecule, the N and C-terminal. strands are closely associated , forming an extended ''tail'', which has some beta-sheet character fo r part of its length and which becomes more disordered towards the ter mini as indicated by N-15{H-1} NOEs. We have considered the structural implications of the possible formation of a native C58-C58 disulphide -bonded homodimer. Comparison of the surface features of pNR-2/pS2 and PSP, and consideration of the sequences of the other human trefoil do mains in the Light of these structures, illuminates the possible role of specific residues in ligand/receptor binding. (C) 1997 Academic Pre ss Limited.