Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants

Abstract

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9⌬C-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9⌬C bound to EGF(AB) H306Y , a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB) H306Y -PCSK9 interaction is pH-independent, LDLR H306Y binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9⌬C containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) 4 has recently emerged as a major regulator of low density lipoprotein (LDL) cholesterol levels in plasma and consequently as an important determinant of cardiovascular health in humans. The link between cardiovascular disease and PCSK9 was initially made following the discovery that the PCSK9 missense mutations, S127R and F216L (1), and later, D374Y (2), are associated with a form of autosomal dominant hypercholesterolemia, a risk factor for coronary heart disease. Subsequently, two PCSK9 nonsense mutations (Y142X and C679X) (3) and several missense mutations (R46L, G106R, N157K, G236S, R237W, L253F, N354I and A443T) (4 -6) have been found to be associated with hypocholesterolemia. Remarkable degrees of protection against coronary heart disease were observed in humans heterozygous for the mutations Y142X and C679X (88% reduced incidence) and by R46L (47% reduced incidence) (7). Consequently, PCSK9 represents a novel therapeutic target for the prevention of premature atherosclerosis and coronary heart disease, and an understanding of its mechanism of action is of significant medical interest.PCSK9 is the ninth member of the mammalian proprotein convertase family of serine proteases. The translated proprotein includes an N-terminal signal sequence directing its secretion (residues 1-30), a prodomain (residues 31-152), a catalytic domain (residues 153-451)...

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation

“…4), in agreement with its reported enhanced LDLR-binding affinity at neutral pH (11,18,23). Concentrations of secreted PCSK9, up to 12 nM (HepG2 cells; ϳ3ϫ endogenous levels; Fig.…”

“…Inhibition of the PCSK9-induced LDLR Degradation by EGF-AB-An EGF-AB peptide containing the LDLR binding site to PCSK9 was reported to inhibit the effect of exogenously added PCSK9 on LDLR degradation in either HEK293 or HepG2 cells (18,23,24). Although the effect of WT EGF-AB on naïve HepG2 cells was not reported, the inhibitory effect of the gain-of-function EGF-AB H306Y is best seen with exogenous PCSK9, with little effect on naïve HepG2 cells (18).…”

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

“…Various approaches include humanized monoclonal antibodies, antisense oligonucleotides, RNA interference, and small molecule inhibitors. Recent success in studies with antibodies directed against PCSK9 has further increased interest in this target (19,20). As progress toward therapeutic intervention of PCSK9-LDLR interactions advances, it is important to understand the molecular basis of the biological events involved.…”

How multi-scale structural biology elucidated context-dependent variability in ectodomain conformation along with the ligand capture and release cycle for LDLR family members

“…In addition, the structure determination of the complex under different pH conditions has suggested that the His-306 of EGF-A forms an additional salt bridge with the Asp-374 of PCSK9 at acidic pH (McNutt et al 2009), which should stabilize the PCSK9-LDLR complex under acidic conditions. In fact, the mutation of His-306 to Tyr is known to be associated with FH (Day et al 1997), and crystallographic analysis has demonstrated that the hydroxyl group of Tyr forms a stable hydrogen bond with Asp-374 even at neutral pH (Bottomley et al 2009;McNutt et al 2009). …”

“…The structure determination of the EGF-AB pair in complex with PCSK9 has shown that the calcium-binding site of EGF-A serves as the binding site for PCSK9 (Bottomley et al 2009;Kwon et al 2008;McNutt et al 2009) (Fig. 3b).…”