Sequence redesign and the assembly mechanism of the oxytocin/bovine neurophysin I biosynthetic precursor

Abstract

The structural organization of neurohypophysial hormone biosynthetic precursors and the interdependence between intramolecular folding and precursor self-association were examined using sequence-engineered mutants of the semisynthetic oxytocin/bovine neurophysin precursor (pros-OT/BNPI). In [N alpha 1-Ac,N epsilon 30,71-diacetimidyl, Ala2,des-His106] Pro-Ot/BNPI or [N alpha 1-Ac,Ala2]pros-OT/BNPI), two structural elements (Tyr2 and free alpha-amino group) were eliminated which were predicted to be critical for intramolecular conformation by stabilizing contact between hormone and neurophysin domains. This mutant was used to test the dependence of precursor self-association on intramolecular conformation. In the second mutant precursor, [N alpha 30,71-diacetimidyl,D-Pro7,D-Leu8,des-His106]p ro-OT/BNPI (or [D-Pro7,D-Leu8]pros-OT/BNPI), the stereochemistry at L-Pro7-L-Leu8 was changed to test the extent to which precursor conformation depends on ordered structure in the processing/spacer sequence which connects the interacting hormone and neurophysin I domains. Intramolecular conformation was characterized for the precursor and mutants by analytical affinity chromatography on immobilized hormone analog Met-Tyr-Phe and by circular dichroism. Data obtained by both methods showed that, while pros-OT/BNPI is folded, with hormone domain occupying the hormone-binding site of the neurophysin domain, the alpha-acetyl-Ala2 mutant is not so organized intramolecularly. When pros-OT/BNPI and the alpha-acetyl-Ala2 mutant were eluted on immobilized BNPII to measure self-association propensity, the native-like precursor was found to bind with 12-15-fold higher affinity than the assembly mutant. Thus, while pros-OT/BNPI assumes a molecular structure containing a high-affinity self-association surface induced by intramolecular hormone domain-neurophysin domain interaction, [N alpha 1-Ac,Ala2]pros-OT/BNPI does not. The results with the alpha-acetyl-Ala2 mutant show that intramolecular domain-domain interaction is the obligatory "trigger" which induces the high-affinity precursor self-association that likely drives precursor to aggregated forms in the concentrated intragranular environment that exists in peptide hormone-synthesizing cells. In contrast, affinity chromatographic and circular dichroism properties of the D-Pro7,D-Leu8 mutant show that this intramolecular trigger is dependent, but only weakly, on the conformation of the peptide sequence between domains, as judged by native-like interaction properties below 40 degrees C but lowered stability to elevated temperature.(ABSTRACT TRUNCATED AT 400 WORDS)