Athetis lepigone is a polyphagous pest found around
the world that feeds on maize, wheat, and various other important
crops. Although it exhibits a degree of resistance to various chemical
insecticides, an effective pest-control method has not yet been developed.
The sex pheromone communication system plays an essential role in
the mating and reproduction of moths, in which pheromone-binding proteins
(PBPs) are crucial genes. In this study, we cloned and purified the
protein AlepPBP1 using an E. coli expression system
and found it had a higher binding affinity to two sex pheromones of A. lepigone, namely, Z7-12:Ac and Z9-14:Ac (with K
i
0.77 ± 0.10 and 1.10
± 0.20 μM, respectively), than to other plant volatiles.
The binding-mode analysis of protein conformation with equilibrium
stabilization was obtained using molecular dynamics (MD) simulation
and indicated that hydrophobic interactions involving several nonpolar
residues were the main driving force for the binding affinity of AlepPBP1
with sex pheromones. Computational alanine scanning (CAS) was performed
to further identify key amino acid residues and validate their binding
contributions. Each key residue, including Phe36, Trp37, Val52, and
Phe118, was subsequently mutated into alanine using site-directed
mutagenesis. Binding assays showed that the efficient binding abilities
to Z7-12:Ac (F36A, W37A, and F118A) and Z9-14:Ac (F36A, W37A, V52A,
and F118A) were almost lost in the mutated proteins. Our results demonstrated
that these key amino acid residues are crucial for determining the
binding ability of AlepPBP1 to sex pheromones. These findings provide
a basis for the use of AlepPBP1 in the studies as a specific target
for the development of novel behavioral antagonists with marked inhibition
or mating-disruption abilities using computer-aided drug design (CADD).