The solution structure of C1-T1, a two-domain proteinase inhibitor derivedfrom a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana a lata (NaProPI) has been expressed and its structure determined by NMR spect roscopy. NaProPI contains six almost identical 53 amino acid repeats that f old into six highly similar domains; however, the sequence repeats do nut c oincide with the structural domains. Five of the structural domains compris e the C-terminal portion of one repeat and the N-terminal portion of the ne xt. The sixth domain contains the C-terminal portion of the sixth repeat an d the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceiv able that adjacent domains in NaProPI interact to generate the circular "br acelet" with the N and C termini in close enough proximity to facilitate fo rmation of the disulphide bonds that form the "clasp" The expressed protein , examined in the current study, comprises residues 25-135 of NaProPI and e ncompasses the first two contiguous structural domains, namely the chymotry psin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue li nker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no n uclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of int eractions between the domains indicates that adjacent domains do not specif ically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from oth er members of the Solanaceae having two, three, or four repeats. The lack o f strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor. (C) 2001 Academic Press.