StAR-like activity and molten globule behavior of StARD6, a male germ-line protein

The steroidogenic acute regulatory protein (StAR) belongs to a family of 15 StAR-related lipid transfer (START) domain proteins termed StARD1-StARD15. StAR (StARD1) induces adrenal and gonadal steroidogenesis by moving cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane by an unclear process that involves conformational changes that have been characterized as a molten globule transition. We expressed, purified, and assessed the activity and cholesterol-binding behavior of StARD1 and StARD3-D7, showing that StARD6 had activity equal to StARD1, whereas StARD4, D5, and D7 had little or no activity with adrenal mitochondria in vitro. Partial proteolysis examined by mass spectrometry suggests that StARD6 has a protease-sensitive C-terminus, similar to but smaller than that of StARD1. Experiments using urea denaturation, stopped-flow kinetics and measurements of mitochondrial membrane association suggests that StARD1 and StARD6 both unfold and refold slowly with similar kinetic patterns. Isothermal titration calorimetry suggests that StARD6 interacts with mitochondrial membranes as well as or better than StARD1. Computational modeling of StARD6 suggests that it has a similar fold to StARD1, with a hydrophobic sterol-binding pocket and a unique C-terminal extension. StARD6, which is expressed only in male germ-line cells, thus exhibits biological and biophysical properties that imply a role in steroidogenesis.