Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin.
The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.
Lin
Ku-Feng
KF
Department of Life Sciences, National Tsing Hua University, Hsinchu,Taiwan.
Lee
Tian-Ren
TR
Tsai
Ping-Hsing
PH
Hsu
Ming-Pin
MP
Chen
Ching-San
CS
Lyu
Ping-Chiang
PC
D001483:Base Sequence; D004261:DNA Replication; D018744:DNA, Plant / Q000235:genetics; D023082:Defensins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D007887:Fabaceae; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism*
D018744:DNA, Plant; D023082:Defensins; D010940:Plant Proteins; D000516:alpha-Amylases