A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30-40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9's prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

Pathogenic gain-of-function mutations in the prodomain and C-terminal domain of PCSK9 inhibit LDL binding.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds and mediates endo-lysosomal degradation of low-density lipoprotein receptor (LDLR), limiting plasma clearance of cholesterol-rich LDL particles in liver. Gain-of-function (GOF) point mutations in PCSK9 are associated with familial hypercholesterolemia (FH). Approximately 30%-40% of PCSK9 in normolipidemic human plasma is bound to LDL particles. We previously reported that an R496W GOF mutation in a region of PCSK9 known as cysteine-histidine-rich domain module 1 (CM1) prevents LDL binding in vitro [Sarkar et al., J. Biol. Chem. 295 (8), 2285-2298 (2020)]. Herein, we identify additional GOF mutations that inhibit LDL association, localized either within CM1 or a surface-exposed region in the PCSK9 prodomain. Notably, LDL binding was nearly abolished by a prodomain S127R GOF mutation, one of the first PCSK9 mutations identified in FH patients. PCSK9 containing alanine or proline substitutions at amino acid position 127 were also defective for LDL binding. LDL inhibited cell surface LDLR binding and degradation induced by exogenous PCSK9-D374Y but had no effect on an S127R-D374Y double mutant form of PCSK9. These studies reveal that multiple FH-associated GOF mutations in two distinct regions of PCSK9 inhibit LDL binding, and that the Ser-127 residue in PCSK9 plays a critical role.