TERTIARY AND QUATERNARY STRUCTURES OF 0.19-ALPHA-AMYLASE INHIBITOR FROM WHEAT KERNEL DETERMINED BY X-RAY-ANALYSIS AT 2.06 ANGSTROM RESOLUTION

The crystal structure of 0.19 alpha-amylase inhibitor (0.19 AI) from w heat kernel was determined by the multiple-isomorphous replacement met hod coupled with density modification and noncrystallographic symmetry averaging and then refined by simulated annealing using diffraction d ata to 2.06 Angstrom resolution (R = 18.7%, free R = 22.3%). The asymm etric unit has four molecules of 0.19 AI, each comprised of 124 amino acid residues. Electron density for residues 1-4 and 69-77 is absent i n all subunits, probably because of the intrinsic flexibility of these segments. Each subunit has four major alpha-helices and one one-turn helix which are arranged in the up-and-down manner, maintaining the fa vorable packing modes of the alpha-helices. 0.19 AI, however, has two short antiparallel beta-strands. All 10 cysteine residues in 0.19 AI f orm disulfide bonds (C6-C52, C20-C41, C28-C83, C42-C99, and C54-C115), consistent with the assignments made biochemically for 0.28 AI from w heat kernel and by NMR analysis of the bifunctional alpha-amylase/tryp sin inhibitor from ragi seeds (RBI). The disulfide bond patterns in th ese AIs are similar to those in the hydrophobic protein from soybean ( HPS), which lack only the bond corresponding to C28-C83 in 0.19 AI. Ex tensive interactions occurred between particular pairs of 0.19 AI subu nits, mainly involving hydrophobic residues. Comparisons of the struct ures of 0.19 AI, RBI, and HPS showed that the arrangements of the majo r alpha-helices are similar but the conformations of the remaining res idues differ markedly. The present X-ray analysis for 0.19 AI and the NMR analysis for RBI suggest that all the AIs in this family have a co mmon fold. The alpha-amylase binding site is discussed on the basis of the tertiary and quaternary structures of 0.19 AI together with bioch emical and spectroscopic data for AIs.