Determination of the Three-Dimensional Structure of the Bifunctional .alpha.-Amylase/Trypsin Inhibitor from Ragi Seeds by NMR Spectroscopy

Abstract

The three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor (RBI) from seeds of ragi (Eleusine coracana Gaertneri) has been determined in solution using multidimensional 1H and 15N NMR spectroscopy. The inhibitor consists of 122 amino acids, with 5 disulfide bridges, and belongs to the plant alpha-amylase/trypsin inhibitor family for which no three-dimensional structures have yet been available. The structure of the inhibitor was determined on the basis of 1131 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 52 phi angles, supplemented by 9 psi and 51 chi 1 angles. RBI consists of a globular four-helix motif with a simple "up-and-down" topology. The helices are between residues 18-29, 37-51, 58-65, and 87-94. A fragment from Val 67 to Ser 69 and Gln 73 to Glu 75 forms an antiparallel beta-sheet. The fold of RBI represents a new motif among the serine proteinase inhibitors. The trypsin binding loop of RBI adopts the "canonical", substrate-like conformation which is highly conserved among serine proteinase inhibitors. The binding loop is stabilized by the two adjacent alpha-helices 1 and 2. This motif is also novel and not found in known structures of serine proteinase inhibitors. The three-dimensional structure of RBI together with biochemical data suggests the location of the alpha-amylase binding site on the face of the molecule opposite to the site of the trypsin binding loop. The RBI fold should be general for all members of the RBI family because conserved residues among the members of the family from the core of the structure.

Determination of the Secondary Structure and Conformation of Puroindolines by Infrared and Raman Spectroscopy

“…Concerning other structural elements, the 3D structures of RBI and wheat ns-LTP clearly reveal lower contents of -sheet or extended structures than those estimated from spectroscopic methods like IR or CD (Strobl et al, 1995;Gincel et al, 1994;Désormeaux et al, 1992;Alagiri & Singh, 1993). The three-dimensional structure of wheat ns-LTP does not reveal the existence of -sheets and shows only 7% -sheets in the case of RBI, while IR and CD predicted a -sheet content of 20-30% for both proteins.…”

“…In this regard, the tryptophan-rich domain (residues 39-45) corresponds to an important extension of the first loops of wheat ns-LTP (Gincel et al, 1994) and of RBI that contain protease inhibitory activity (Strobl et al, 1995).…”

“…Few three-dimensional (3D) structures are known for these proteins. Recently, Strobl et al (1995) have determined the 3D structure of a bifunctional R-amylase/trypsin inhibitor from ragi seeds (RBI) by NMR spectroscopy. This protein, which is homologous to the monomeric and homodimeric R-amylase inhibitor from wheat seeds, is comparable in size to puroindolines with about 120 amino acid residues.…”

Disulphide structure of a sunflower seed albumin: conserved and variant disulphide bonds in the cereal prolamin superfamily

“…3), and a bifunctional c~-amylase/ trypsin inhibitor (RBI) from ragi (Indian finger millet) [14] (not shown). Again it is possible to identify conserved cysteine residues homologous to those present in the 2S albumins and the gliadins, but the patterns of disulphide bond formation are not identical.…”

Multiple rotation function

“…This protein crystallizes in space group P4 2 2 1 2, with unit-cell parameters a = b = 57.12, c = 80.24 A Ê . A joint analysis of the rotation functions calculated by AMoRe in the resolution range 4±15 A Ê with all 20 NMR models taken from the PDB (Strobl et al, 1995) gives an extremely strong signal corresponding to the correct solution (Figs. 2c and 2d) when D min varies between 2 and 4 .…”