Regulation of LDL-Cholesterol by Circulating PCSK9 and its Natural Variants

Abstract

Circulating proprotein convertase subtilisin/kexin type-9 (PCSK9) acts as a binding chaperone to mediate endo-lysosomal degradation of cell surface low-density lipoprotein receptor (LDLR). Gain-of-function (GOF) mutations in PCSK9 are causative for autosomal dominant hypercholesterolemia (ADH). An acidic intrinsically disordered region (IDR) in the N-terminus of PCSK9's prodomain mediates autoinhibition of LDLR binding, possibly involving an electrostatic intramolecular interaction. Our lab previously showed that contact with hydrophobic membranes triggers the formation of a transient amphipathic-helix within the IDR required for high-affinity binding of PCSK9 to LDL particles, which antagonizes the PCSK9-LDLR interaction. In a recent collaboration using hydrogen-deuterium exchange/mass spectrometry analysis, we found evidence that the IDR interacts in an aqueous environment with a helical region of the prodomain enriched in positive-charged arginine residues. Interestingly, this region was previously shown to bind heparin sulfate proteoglycans (HSPGs) on the surface of hepatocytes to facilitate the PCSK9-LDLR interaction. An ADH-associated R105W GOF mutation within this helix significantly lowered LDL binding in vitro, as did a Y107F substitution to disrupt an intramolecular bond with Asp-129 in an adjacent helix. Multiple amino acid substitutions for Asp-129 abolished PCSK9-LDL binding, including two ADH-associated GOF mutations (D129G and D129N). Compared to wild-type PCSK9, a PCSK9-D129A variant displayed >3-fold increased binding to purified LDLR extracellular domain and enhanced ability to mediate cell surface LDLR degradation in mouse Hepa1c1c7 hepatoma cells. Thus, like the IDR, the Asp-129 is autoinhibitory of the PCSK9-LDLR interaction. This work supports a model whereby PCSK9's N-terminal acidic IDR contacts basic patches in the prodomain or C-terminal domain under aqueous versus hydrophobic conditions, respectively, with the latter allowing LDL to bind at or near Asp-129. Consequently, LDL binding would mimic the allosteric autoinhibitory effect of the IDR and antagonize the PCSK9-LDLR interaction. Conversely, the binding of PCSK9 to cell surface HSPGs would displace the IDR and mitigate its autoinhibitory effect, thereby promoting PCSK9-LDLR binding and subsequent LDLR degradation in hepatocytes.