Insect Control: Biological and Synthetic Agents - Index of

128 4: Insect Growth- and Development-Disrupting Insecticides
Figure 3 Different elevations of computer model(s) of the three-dimensional structure of the LBD of insect ecdysone receptors.
The different helices are represented in different colors, and indicated by arrows and numbers. After a ligand has docked into the
ligand binding pocket of EcR heterodimerizd with USP, helix 12 closes on the binding cavity like a ‘‘mouse trap.’’
ability to bind was unaffected. Changing A110 to
leucine was as effective as the change to proline.
Further work along these lines should make it possible
not only to design new effective chemistries as
ecdysone agonists/antagonists for pest control, but
also to design EcR LBDs that result in productive
binding with different chemistries, thus allowing
their use in several gene switch applications.
The necessity of EcR and USP forming a heterodimer
for ligand interaction coupled with the difficulty
in producing large enough quantities of EcR
and USP LBDs required for X-ray diffraction studies
have been overcome by Billas et al. (2003). These
authors reported the crystal structures of the LBDs
of the moth H. virescens EcR-USP heterodimer in
complex with the ecdysteroid ponasterone A and
with a nonsteroidal, lepidopteran specific agonist,
BY106830 (a bisacylhydrazine). Comparison of the
crystal structures liganded with ponasterone A and
BY106830 revealed that the two ligands occupy
very different but slightly overlapping spaces in the
ligand binding pockets. The overlap of the two
ligands was observed on the side chain of ponasterone
A with the t-butyl group and the benzoyl ring
closest to it in BY106830. The presence of the
t-butyl group and its occupation in a hydrophobic
groove of the HvEcR LB pocket confers lepidopteran
specificity on it. Further examination of the residues
in this hydrophobic cavity revealed that V384
in helix 5, which was conserved in lepidopteran insect
EcR LBDs and is replaced by methionine in other
insects, is essential for the lepidopteran specificity.
The crystal structure observations supported the
results of Kumar et al.(2002).
4.2.2.3. Ligand-dependent conformational
changes Yao et al. (1995) used limited proteolysis
protection to demonstrate that binding of muristerone
A, a potent ecdysteroid, to Drosophila melanogaster
EcR and USP (DmEcR/DmUSP) induces a
conformation change in EcR that can be detected
by sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS–PAGE) and autoradiography. In
these experiments, incubation of muristerone
A with DmEcR- 35 S-methionine labeled/DmUSP
proteins produced by transcription and translation
of corresponding cDNAs in rabbit reticulocyte cellfree
mixtures, afforded partial protection to EcR
from trypsin as the protease. In an extension of this
approach, Dhadialla et al. (unpublished data) used
EcR- 35 S-methionine labeled/USP proteins produced
in vitro to demonstrate that muristerone A bound to
DmEcR/DmUSP, Aedes aegypti EcR and USP
(AeEcR/AeUSP), and CfEcR/CfUSP protected an
EcR fragment of 36 kDa from limited proteolysis
with trypsin, chymotrypsin, and proteinase K. On
the other hand, the ability of tebufenozide (RH-
5992) to induce a similar conformational change
upon binding to EcR, which affords limited proteolytic
protection as with muristerone A, correlated
with its affinity to the various EcRs (Table 3). Interestingly,
peptide fragments of a similar size (36 kDa)