PDBsum entry 3gcw

Protein binding

PDB id: 3gcw

Contents

Protein chains

92 a.a. *

434 a.a. *

41 a.a. *

Metals

_CA

* Residue conservation analysis

PDB id:

3gcw

Name:

Protein binding

Title:

Pcsk9:egfa(h306y)

Structure:

Proprotein convertase subtilisin/kexin type 9. Chain: p. Synonym: proprotein convertase pc9, subtilisin/kexin-like protease pc9, neural apoptosis-regulated convertase 1, narc-1. Engineered: yes. Proprotein convertase subtilisin/kexin type 9. Chain: a. Synonym: proprotein convertase pc9, subtilisin/kexin-like protease pc9, neural apoptosis-regulated convertase 1, narc-1.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: pcsk9, narc1, psec0052. Expressed in: homo sapiens. Expression_system_taxid: 9606. Expression_system_cell_line: human embryonic kidney cells (hek293s). Gene: ldlr. Expressed in: escherichia coli.

Resolution:

2.70Å R-factor: 0.236 R-free: 0.278

Authors:

H.J.Kwon

Key ref:

M.C.McNutt et al. (2009). Antagonism of Secreted PCSK9 Increases Low Density Lipoprotein Receptor Expression in HepG2 Cells. J Biol Chem, 284, 10561-10570. PubMed id: 19224862 DOI: 10.1074/jbc.M808802200

Date:

22-Feb-09 Release date: 03-Mar-09

Protein chain

Q8NBP7  (PCSK9_HUMAN) -  Proprotein convertase subtilisin/kexin type 9 from Homo sapiens

Seq: 692 a.a.

Struc: 92 a.a.

Protein chain

Q8NBP7  (PCSK9_HUMAN) -  Proprotein convertase subtilisin/kexin type 9 from Homo sapiens

Seq: 692 a.a.

Struc: 434 a.a.*

Protein chain

P01130  (LDLR_HUMAN) -  Low-density lipoprotein receptor from Homo sapiens

Seq: 860 a.a.

Struc: 41 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains P, A: E.C.3.4.21.- - ?????

DOI no: 10.1074/jbc.M808802200

J Biol Chem 284:10561-10570 (2009)

PubMed id: 19224862

Antagonism of Secreted PCSK9 Increases Low Density Lipoprotein Receptor Expression in HepG2 Cells.

M.C.McNutt, H.J.Kwon, C.Chen, J.R.Chen, J.D.Horton, T.A.Lagace.

ABSTRACT

PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

Selected figure(s)

Figure 2.
Structure of the PCSK9-EGF-A complex. The sigmaA weighted 2F[o] - F[c] electron density map contoured at 1σ shows the conformational change that occurs upon protonation of His-306. EGF-A and PCSK9 are represented as a stick model. Residues involved in the pH-dependent conformational change are colored according to element type as follows: nitrogen, blue; oxygen, red; EGF-A carbon, yellow; PCSK9 carbon, green. All other residues are colored gray. A, at acidic pH, His-306 of EGF-A forms a salt bridge with Asp-374 of PCSK9. B, at neutral pH, His-306 of EGF-A forms an intramolecular hydrogen bond with Ser-305. C, FH mutation H306Y of EGF-A is able to form a hydrogen bond with Asp-374 of PCSK9 at neutral pH.

Figure 3.
EGF-AB(H306Y) blocks uptake of PCSK9 in HuH7 cells. HuH7 cells were treated 30 min with 5 μg/ml purified full-length FLAG-tagged PCSK9 alone or in combination with 5 μm EGF-AB(H306Y) blocking peptide or EGF-AB(L318A) negative-control peptide. A–D, cells were immunostained for PCSK9 (green) and nuclear stained with 4′,6-diamidino-2-phenylindole (magenta) as described under “Experimental Procedures.” E, cells were lysed and subjected to SDS-PAGE and immunoblotting for FLAG-tagged PCSK9 as described in the legend to Fig. 1B. Actin was detected as a control for equal protein loading. PCSK9 and actin levels were visualized and quantified using an IRDye800-labeled secondary antibody and the LI-COR Odyssey infrared imaging system. PCSK9 levels were normalized to actin expression.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 10561-10570) copyright 2009.

Figures were selected by an automated process.