A Two-step Binding Model of PCSK9 Interaction with the Low Density Lipoprotein Receptor

Abstract

PCSK9 (proprotein convertase subtilisin-like/kexin type 9) is an emerging target for pharmaceutical intervention. This multidomain protein interacts with the LDL receptor (LDLR), promoting receptor degradation. Insofar as PCSK9 inhibition induces a decrease in plasma cholesterol levels, understanding the nature of the binding interaction between PCSK9 and the LDLR is of critical importance. In this study, the ability of PCSK9 to compete with apoE3 N-terminal domain-containing reconstituted HDL for receptor binding was examined. Whereas full-length PCSK9 was an effective competitor, the N-terminal domain (composed of the prodomain and catalytic domain) was not. Surprisingly, the C-terminal domain (CT domain) of PCSK9 was able to compete. Using a direct binding interaction assay, we show that the PCSK9 CT domain bound to the LDLR in a calcium-dependent manner and that co-incubation with the prodomain and catalytic domain had no effect on this binding. To further characterize this interaction, two LDLR fragments, the classical ligand-binding domain (LBD) and the EGF precursor homology domain, were expressed in stably transfected HEK 293 cells and isolated. Binding assays showed that the PCSK9 CT domain bound to the LBD at pH 5.4. Thus, CT domain interaction with the LBD of the LDLR at endosomal pH constitutes a second step in the PCSK9-mediated LDLR binding that leads to receptor degradation.

Novel Domain Interaction Regulates Secretion of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Protein

“…Our results are in agreement with recent findings reported by Yamamoto et al (31) showing that CD is involved in the PCSK9-LDLR interaction on the cell surface.…”

Recent advances in physiological lipoprotein metabolism

“…A second binding site between the positively charged C-terminal domain of PCSK9 and the negatively charged ligand binding site of the LDL receptor with a much greater binding at pH 5.4 has been identified. This constitutes a second step driving the LDL receptor towards degradation [146,147]. Association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDL receptors, blunting PCSK9-mediated LDL receptor degradation.…”

PCSK9 regulates neuronal apoptosis by adjusting ApoER2 levels and signaling

“…Unlike the other PCs, the proteolytic function of PCSK9 is autoinhibited by non-covalent binding of the pro-domain to the catalytic site [5]. PCSK9 binds to the extracellular EGF-A repeat of low-density lipid receptor family members lowdensity lipoprotein receptor (LDLR), very low-density lipoprotein receptor (VLDLR), and apolipoprotein receptor-2 (ApoER2) [6,7], and targets them for degradation in the intracellular acidic compartments [8]. Gain-and loss-of-function mutants of PCSK9 are associated with hyper-and hypocholesterolemia, respectively, by affecting systemic levels of LDLR and cholesterol uptake [9].…”