KEY DISULFIDE BONDS IN AN INSECT HORMONE-BINDING PROTEIN - CDNA CLONING OF A JUVENILE-HORMONE BINDING-PROTEIN OF HELIOTHIS-VIRESCENS AND LIGAND-BINDING BY NATIVE AND MUTANT FORMS

The hemolymph juvenile hormone binding protein (JHBP) from the early f ifth instar larvae of Heliothis virescens (Lepidoptera, Noctuidae) has been purified, and three cDNA clones for this protein have been isola ted from a fat body cDNA Library constructed in bacteriophage lambda Z AP XR. The deduced amino acid sequence of the full-length clone predic ts a mature protein consisting of 224 residues, a molecular mass of 24 976 Da, and a pI of 5.29. Comparison of the amino acid sequence to th at of the previously described JHBP from Manduca sexta shows 51% overa ll identity with highly conserved N-and C-terminal regions. One of the three clones bound photoactivatable analogs of juvenile hormones with much lower affinity than the other two. This clone had Phe(150) in pl ace of the expected Cys(150) conserved in other JHBP clones. The F150C mutant of this clone regained native binding affinity. For native Hvi r-JHBP, the affinity for [H-3]JH I was lower under reducing conditions (87 nM) relative to a 40 nM affinity under nonreducing conditions. Th e importance of pairs of Cys residues was addressed by preparing Cys t o Ala mutants at each site. Expressed proteins were tested for binding affinity by photoaffinity labeling with tritium-labeled JH analogs an d by binding assays using (10R,11S)-[H-3]JH I. Curiously, the C150A mu tant retained full activity, implying that the aberrant C150F was dysf unctional due to steric hindrance rather than to a missing disulfide L inkage. Likewise, C29A and C194A had binding affinities unchanged from that of the full-length wild-type clone. Two constructs, C9A and C16A , showed reduced binding affinity, suggesting that they form a disulfi de bond important for ligand recognition.