THE CRYSTAL STRUCTURE OF A BI-FUNCTIONAL KUNITZ TYPE CYSTEINE PROTEINASE INHIBITOR AT 1.9Å RESOLUTION. Minakshi Ghosh1, Michael Mares2, Karl Harlos1 and Colin Blake1. 1Laboratory of Molecular Biophysics, Rex Richards Building, South Parks Road, Oxford OX1 3QU, UK; 2Institute of Organic Chemistry and Biochemistry, CSAV, Flemingovo namesti 2, Prague, Czech Republic.
The cysteine proteinase inhibitor from potato (PCI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and shows inhibitory properties against a broad range of serine proteinases too. It consists of 180 amino acid residues. The primary sequence of PCI shows about 20% sequence homology with the other proteinase inhibitors of the family.
The crystal structure of PCI has been determined to 1.9Å by the method of single isomorphous replacement and refined using the program XPLOR. The current model includes 179 amino acid residues and 107 water molecules, with a rms deviation of 0.008Å in bond lengths and 1.6* in bond angles from identity. The crystallographic R-factor is 21.1% (Free R-factor = 25.6%) including all reflections from 8Å-1.9Å. The three-dimensional structure of PCI shows the characteristic [[beta]]-trefoil fold of the STI Kunitz inhibitor family. The core of the structure, consisting of 12 antiparallel [[beta]]-sheets are similar to ETI, the trypsin inhibitor from Erythrina caffra seeds, while the long loops connecting the [[beta]]-strands are considerably different in length and fold. The reactive sites of the other inhibitors for trypsin and those of a bi-functional inhibitor of proteinase K (which is similar to the bacterial subtilisin) and [[alpha]]-amylase, belonging to the same family, are all seen to be located on these loops. The position of the active site of PCI is not known yet but it is likely that this is also located on one of these loops. Identification of the reaction centres is in progress and is expected to reveal how, on a common structural framework, different reaction centres for different proteinases are accommodated.