David J. Craik, Katherine J. Nielsen, Robyn L. Heath+, Marcus Lee*, and
Marilyn A. Anderson*
Centre for Drug Design and Development, University of Queensland, St Lucia QLD 4072, Australia, and *Department of Biochemistry and +Department of Agriculture, La Trobe University, Bundoora VIC 3083, Australia.
Plants contain many interesting proteins which have potential pharmaceutical applications. NMR is a powerful technique for determining structures of these proteins, as will be illustrated by examples of several plant proteins recently studied in our laboratory. The stigmas of N. alata are rich in proteinase inhibitors (PIs), which are probably involved in the protection of the female reproductive organs against insect damage. The PIs are derived from a highly expressed precursor protein of 40.3 kDa which is processed proteolytically during flower development to release five homologous inhibitors of ~6 kDa, one of which inhibits chymotrypsin and four inhibit trypsin (Atkinson et al., 1993). The three-dimensional structures of the chymotrypsin inhibitor C1 and the trypsin inhibitor T1 were solved by NMR spectroscopy (Nielsen et al., 1994). Both structures are well defined and contain a triple-stranded [[beta]]-sheet as the dominant secondary structural feature. Several turns and a short region of 310 helix are also present. The putative chymotrypsin-reactive site is present on an exposed loop which is less defined than the rest of the protein. The overall shape of both proteins is disc-like and the N- and C-termini are exposed, supporting the proposal that they result from post-translational processing of the 40.3 kDa precursor protein. The disc-like structure of C1 and T1 and the distribution of their hydrophobic residues led to the speculation that the individual inhibitors are stacked in the precursor. Modelling studies on the structure of the combined domains will be described. Studies on a protein derived from the African plant O. affinis will also be described. The protein, kalata, is a potent uterotonic agent, first discovered because of its use in native medicine as an aid in accelerating labour. The NMR-derived structures show that the protein contains a cystine-knot motif in which one disulfide bond penetrates an embedded loop in the protein formed by two other disulfide bonds and the peptide fragments joining them. This unique structural feature is thought to be responsible for the exceptional stability of this protein.
1. Atkinson, A.H., Heath, R.L., Simpson, R.L., Clarke, A.C., & Anderson, M.A. (1993) Proteinase inhibitors in Nicotiana alata are derived from a precursor protein which is expressed into five homologous inhibitors. Plant Cell 5, 203-213.
2. Nielsen, K.J., Heath, R.L., Anderson, M.A., & Craik, D.J. (1994) The three-dimensional solution structure by 1H NMR of a 6 kDa proteinase inhibitor isolated from the stigma of Nicotiana alata. J. Mol. Biol. 242, 231-243.