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Updated: Jun 28, 2026
A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
Published on: August 28, 2018
Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial
Matthew J Bottomley1, Agostino Cirillo, Laura Orsatti
1Department of Biochemistry, Istituto di Ricerca di Biologia Molecolare "P. Angeletti", Via Pontina Km 30.600, 00040 Pomezia (Rome), Italy. matthew_bottomley@merck.com
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 PCSK9DeltaC-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 PCSK9DeltaC 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 PCSK9DeltaC 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.
Insights
Proprotein convertase subtilisin/kexin type 9 (PCSK9) interaction with the low-density lipoprotein receptor (LDLR) is pH-dependent. Mutations in LDLR associated with familial hypercholesterolemia (FH) alter PCSK9 binding, impacting LDL uptake.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein receptor (LDLR) levels, making it a therapeutic target for atherosclerosis and coronary heart disease.
- Understanding the molecular interactions between PCSK9 and LDLR is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the pH-dependent interaction between PCSK9 and the Epidermal Growth Factor (EGF) repeats of LDLR.
- To elucidate the structural basis of PCSK9-LDLR binding and the impact of familial hypercholesterolemia (FH)-associated mutations.
Main Methods:
- X-ray crystallography was used to determine the structures of PCSK9 complexes with LDLR EGF repeats at neutral and low pH.
- Site-directed mutagenesis was employed to study the effects of specific mutations on PCSK9-LDLR binding affinity and pH dependence.
- Cellular assays were performed to assess the functional consequences of these interactions on LDL uptake.
Main Results:
- PCSK9 binds LDLR EGF(AB) repeats in a pH-dependent manner, requiring PCSK9 autocleavage but not its C-terminal domain.
- Structural analysis revealed rearrangements in the EGF(A) His-306 and its interaction with PCSK9 Asp-374, explaining enhanced binding at low pH.
- FH-associated mutations (H306Y, D374Y, D374H) in LDLR alter PCSK9 binding affinity and pH dependence, providing a molecular rationale for disease phenotypes.
Conclusions:
- The study provides detailed structural insights into the pH-dependent mechanism of PCSK9-LDLR interaction.
- Specific LDLR mutations associated with FH significantly modulate PCSK9 binding, affecting LDL metabolism.
- These findings offer a basis for designing novel therapeutic strategies targeting PCSK9-LDLR interactions for cardiovascular disease prevention.
