Refined crystal structure (2.3 angstrom) of a double-headed winged bean alpha-chymotrypsin inhibitor and location of its second reactive site

The crystal structure of a double-headed alpha-chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 Angstrom resolution to an R-factor of 18.7% for 9,897 reflections. The crystals belong to the hexa gonal space group P6(1)22 with cell parameters a = b = 61.8 Angstrom and c = 212.8 Angstrom. The final model has a good stereochemistry and a root mea n square deviation of 0.011 Angstrom and 1.14 degrees from ideality for bon d length and bond angles, respectively. A total of 109 ordered solvent mole cules were localized in the structure. This improved structure at 2.3 Angst rom led to an understanding of the mechanism of inhibition of the protein a gainst alpha-chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the p rotein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve antiparallel beta-strands w ith connecting loops arranged in a characteristic beta-trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family a s well as to some non homologous functionally unrelated proteins. A wide va riation in the surface loop regions is seen in the latter ones. (C) 1999 Wi ley-Liss, Inc.