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

Abstract

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.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway

“…However, we found that the anti-PCSK9 antibody alirocumab failed to regulate AKT signaling. Alirocumab has been shown to bind the catalytic domain and prodomain of PCSK9, thereby blocking its interaction with LDLR (Hess et al, 2018;Sarkar et al, 2020), while PCSK9 palmitoylation occurred at the C600 site, not the catalytic domain nor prodomain of PCSK9; therefore, alirocumab failed to inhibit PI3K-AKT signaling pathway activation or attenuate sorafenib resistance. Based on these findings, we developed a biologically active PCSK9-derived peptide that competitively binds endogenous PCSK9 and inhibits its palmitoylation, thereby blocking AKT phosphorylation and enhancing the antitumor effects of sorafenib in HCC.…”

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

“…In the case of PCSK9, it was recently shown that the N-terminal acidic IDR can adopt transient α-helical structure (Ultsch et al, 2019;Sarkar et al, 2020). Computational modeling supported this structural shift aligns an intramolecular interaction with CM1, which is enriched in basic residues (Sarkar et al, 2020).…”

“…Frontiers in Physiology frontiersin.org critical to LDL binding, namely an N-terminal IDR in the prodomain and CM1 in the CHR domain (Kosenko et al, 2013;Sarkar et al, 2020). We previously identified several GOF mutations in CM1 that confer lowered (R469W and F515L) or abolished (R496W) LDL binding ability in vitro (Sarkar et al, 2020). Although distant from these residues, several GOF mutations in the prodomain (L108R, S127R, and D129G) are localized on approximately the same molecular plane (Figure 2A).…”

“…Reaction mixtures were separated on agarose gels (top) and fluorophore-labeled PCSK9 binding to LDL was quantified and fitted to competition binding curves using non-linear regression (n = 3) (bottom). Frontiers in Physiology frontiersin.org critical to LDL binding, namely an N-terminal IDR in the prodomain and CM1 in the CHR domain (Kosenko et al, 2013;Sarkar et al, 2020). We previously identified several GOF mutations in CM1 that confer lowered (R469W and F515L) or abolished (R496W) LDL binding ability in vitro (Sarkar et al, 2020).…”

Association of PCSK9 with human plasma Lipoproteins

“…However, it has been suggested that the above correlation could also be attributed to the association of PCSK9 with LDL particles in the circulation. Indeed, studies have demonstrated that approximately 30% to 40% of plasma PCSK9 is associated with LDL through a protein-protein interaction with its ApoB-100 content 36 (figure 1) and there is evidence that this association occurs within the secretory pathways of hepatocytes 37 . Moreover, the PCSK9 interaction with ApoB containing lipoproteins leads to the suppression of the ApoB degradation through the autophagosome/lysosome pathway 37 .…”