Molecular basis for LDL receptor recognition by PCSK9

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally regulates hepatic low-density lipoprotein receptors (LDLRs) by binding to LDLRs on the cell surface, leading to their degradation. The binding site of PCSK9 has been localized to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. Here, we describe the crystal structure of a complex between PCSK9 and the EGF-A domain of the LDLR. The binding site for the LDLR EGF-A domain resides on the surface of PCSK9's subtilisin-like catalytic domain containing Asp-374, a residue for which a gain-of-function mutation (Asp-374-Tyr) increases the affinity of PCSK9 toward LDLR and increases plasma LDL-cholesterol (LDL-C) levels in humans. The binding surface on PCSK9 is distant from its catalytic site, and the EGF-A domain makes no contact with either the C-terminal domain or the prodomain. Point mutations in PCSK9 that altered key residues contributing to EGF-A binding (Arg-194 and Phe-379) greatly diminished binding to the LDLR's extracellular domain. The structure of PCSK9 in complex with the LDLR EGF-A domain defines potential therapeutic target sites for blocking agents that could interfere with this interaction in vivo, thereby increasing LDLR function and reducing plasma LDL-C levels.

Fig. 1.

The PCSK9:EGF-A complex. (A) PCSK9, with the prodomain (magenta), the subtilisin-like catalytic domain (green), and the C-terminal domain (brown), and the EGF-A domain of LDLR (blue) is represented as a ribbon diagram. The bound calcium ion within the EGF-A domain is shown as a red sphere. (B) Superposition of the PCSK9:EGF-A complex and apo-PCSK9. The PCSK9:EGF-A complex is shown with PCSK9 in green, EGF-A in blue, and bound calcium as a red sphere. Apo-PCSK9 is shown in red. (C) The apo-PCSK9 (Protein Data Bank ID code 2P4E, blue; Protein Data Bank ID code 2PMW, cyan; Protein Data Bank ID code 2QTW, magenta) structures superimposed onto the PCSK9:EGF-A complex [carbon, gray (PCSK9) or yellow (EGF-A); nitrogen, blue; oxygen, red]. Arg-194 from PCSK9 forms a salt bridge with Asp-310 of EGF-A, breaking an intramolecular salt bridge with Glu-197.

Fig. 2.

Interface between PCSK9 and EGF-A. (A) The surface of PCSK9 buried upon binding of EGF-A. The surface of PCSK9 buried upon binding to EGF-A is colored according to element type: carbon, orange; nitrogen, blue; oxygen, red; sulfur, green. Areas of PCSK9 not involved in binding are colored gray. EGF-A is represented as a stick model. Residues within EGF-A involved in binding are colored according to element type: carbon, yellow; nitrogen, blue; oxygen, red. Residues not involved in binding are colored gray. (B) Interactions between PCSK9 (gray) and EGF-A (yellow) near the calcium (green sphere) binding site of EGF-A. (C) Autocatalysis of PCSK9 is required for binding to EGF-A. Autocatalysis of PCSK9 (gray) between residues 152–153 results in a free N-terminal amine that interacts with EGF-A (yellow). The site of the gain-of-function mutation, Asp-374, on PCSK9 is positioned to interact with His-306 of EGF-A. (D) Calcium coordination within the EGF-A domain after binding to PCSK9. The sigmaA weighted, 2F_o − F_c electron density map contoured at 1σ shows the calcium ligands arranged as a pentagonal bipyramid. Asp-310 coordinates calcium (green sphere) and forms a salt bridge with Arg-194 of PCSK9. A water molecule (red sphere) acts as an additional ligand.

Fig. 3.

Mutations in PCSK9 and LDLR. (A) The gain-of-function mutation Asp-374–Tyr in PCSK9 increases binding to LDLR. Asp-374 in PCSK9 modeled as tyrosine (gray) is in position to hydrogen bond to His-306 of LDLR. (B) The FH mutation His-306–Tyr in LDLR. His-306 in LDLR modeled as tyrosine (gray) is in position to hydrogen bond to Asp-374 of PCSK9. (C) Model for full-length LDLR-ECD bound to PCSK9. The EGF-A domain (blue) of the LDLR-ECD (cyan) at acidic pH and the PCKSK9:EGF-A complex were superimposed. PCSK9 (prodomain, magenta; subtilisin-like catalytic domain, green; C-terminal domain, brown) binds on the outside surface of LDLR and would not interfere with the interaction of ligand binding modules R4 and R5 with the β-propeller domain.