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.