Juvenile hormone receptor relies on conserved domain interfaces for dimerization-dependent activity.

Juvenile hormone (JH) is vital to insects. In Drosophila, JH signals through a basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) protein methoprene-tolerant (MET) and its paralog germ cell-expressed (GCE). Binding of JH to the PAS-B domain of MET or GCE triggers the assembly of a DNA-bound, transcriptionally active JH receptor (JHR) heterodimer comprising either MET or GCE and their partner taiman (TAI). Molecular mechanisms of the JHR complex activation remain unclear. Here, we use the Drosophila GCE protein to address the presumably critical but as yet unexplored function of the other PAS domain (PAS-A) in JHR signaling. We show that charged amino acid residues, highly conserved within the PAS-A domain across insect and mammalian bHLH-PAS proteins, are required for JHR signaling in vitro and in vivo. Mutations at these sites do not affect the ability of GCE to bind JH to its PAS-B domain. However, they compromise the JH-induced nuclear import of GCE, its dissociation from the chaperone heat-shock protein HSP83, and dimerization with TAI, consequently preventing the DNA binding and transcriptional JHR activities. Functionality of the mutated GCE variants in developing flies is also impaired. Molecular modeling suggests that mutations of the charged GCE PAS-A residues alter the natural salt bridges critical for intra- and interdomain interactions. This in turn causes non-native configurations of both GCE PAS domains, making them incompatible with TAI dimerization and possibly augmenting GCE attachment to HSP83. These results suggest that JHR signaling critically depends on interactions between PAS-A and PAS-B domains which are allosterically coupled.