STRUCTURAL STUDIES OF DETERGENT-SOLUBILIZED AND VESICLE-RECONSTITUTEDLOW-DENSITY-LIPOPROTEIN (LDL) RECEPTOR

The low-density lipoprotein (LDL) receptor plays a key role in maintai ning circulating and cellular cholesterol homeostasis. The LDL recepto r is a transmembrane glycoprotein whose biochemical and genetic proper ties have been extensively studied notably by Brown, Goldstein and col leagues [Brown, M. S., & Goldstein, J. L., (1986) Science 232, 34-47]. However, few if any structural studies of the LDL receptor have been reported, and details of its secondary and tertiary structure are lack ing. In an attempt to determine the low-resolution structure of the LD L receptor, we have purified the receptor from bovine adrenal cortices using modifications of the method of Schneider et al. [Schneider, W. J., Goldstein, J. L., & Brown, M. S. (1985) Methods in Enzymol. 109, 4 05-417]. Using circular dichroism, the secondary structure of the dete rgent-solubilized bovine LDL receptor at 25 degrees C was shown to be 19% alpha-helix, 42% beta-sheet, and 39% random coil. Interestingly, t he detergent-solubilized receptor appeared to be quite resistant to ch anges in secondary structure over the temperature range 10-90 degrees C, with only minor but reversible changes being observed. In contrast, a more pronounced unfolding of the deter ent-solubilized receptor was observed in the presence of guanidinium hydrochloride. Using the comp lete sequence of the human LDL receptor, sequence analysis by the Chou -Fasman prediction algorithm showed quite good agreement with the expe rimentally determined secondary structure of the bovine LDL receptor a t 25 degrees C. Finally, the purified, bovine LDL receptor was reconst ituted into large unilamellar vesicles of egg yolk phosphatidylcholine using a procedure exploiting preformed vesicles and detergent dialysi s. We showed previously using negative stain electron microscopy that reconstituted vesicles bind LDL. Now, using cryoelectron microscopy of frozen hydrated reconstituted vesicles evidence of an extended, stick -like morphology (length similar to 120 Angstrom) for the extracellula r domain of the LDL receptor has been obtained. Successful purificatio n of the receptor, its incorporation into single bilayer vesicles, and its direct visualization by cryoelectron microscopy pave the way for more detailed structural studies of the LDL receptor and the receptor- LDL complex.