Structure‐based protein engineering for α‐amylase inhibitory activity of plant defensin

Abstract

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.

Flower-specific jasmonate signaling regulates constitutive floral defenses in wild tobacco

“…S9B). Structural studies of the N. alata floral defensin NaD1 indicated that it contains an α-amylase inhibitory activity region that was similar to the one found in VrD1, an insecticidal defensin in Vigna radiata (64,65). Because N. alata NaD1 has high sequence similarity with NaDEF1 (80%) and NaDEF2 (100%), we inferred that these floral defensins in N. attenuata might also have insecticidal properties (SI Appendix, Fig.…”

Development and Engineering of CSαβ Motif for Biomedical Application

“…Compared with single-domain antibodies, the proteins with a CSαβ motif have smaller molecular weights and higher structural stabilities (Holt et al, 2003;Skerra, 2007;Yang et al, 2009). As previously described, proteins with a CSαβ motif share low sequence identity but high structural similarity (Lin et al, 2007). Their functions are highly varied and sturctures are ultra-stable.…”

“…For example, loop 1 of the Arabidopsis thaliana trypsin inhibitor (ATT p ) and loop 2 of cowpea thionine are the functional epitopes for trypsin inhibition (Wijaya et al, 2000;Zhao et al, 2002). The loop 3 of Vigna radiate defensin 1 (VrD1) is the functional loop for insect α-amylase inhibition (Lin et al, 2007;Liu et al, 2006). Fig.…”

“…Based on the scaffold, some CSαβ proteins have been engineered to exhibit new functions or changing of antimicrobial activities (Lin et al, 2007;Vita et al, 1999;Vita et al, 1995). Different approaches, including minimal residue substitution, functional epitope exchange, structural based modification and combinatorial chemistry have been employed to engineer the scaffold to exhibit new functions (Lin et al, 2007;Thevissen et al, 2007;Van Gaal et al, 2004;Vita et al, 1999;Vita et al, 1995;Zhao et al, 2004).…”

“…Different approaches, including minimal residue substitution, functional epitope exchange, structural based modification and combinatorial chemistry have been employed to engineer the scaffold to exhibit new functions (Lin et al, 2007;Thevissen et al, 2007;Van Gaal et al, 2004;Vita et al, 1999;Vita et al, 1995;Zhao et al, 2004). Protein structure is important to the rational design of a protein scaffold.…”