spacer
spacer

PDBsum entry 3gcw

Go to PDB code: 
protein metals Protein-protein interface(s) links
Protein binding PDB id
3gcw
Jmol
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 p 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 p 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 ( 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    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q8NBP7  (PCSK9_HUMAN) -  Proprotein convertase subtilisin/kexin type 9
Seq:
Struc:
 
Seq:
Struc:
692 a.a.
92 a.a.
Protein chain
Pfam   ArchSchema ?
Q8NBP7  (PCSK9_HUMAN) -  Proprotein convertase subtilisin/kexin type 9
Seq:
Struc:
 
Seq:
Struc:
692 a.a.
434 a.a.*
Protein chain
Pfam   ArchSchema ?
P01130  (LDLR_HUMAN) -  Low-density lipoprotein receptor
Seq:
Struc:
 
Seq:
Struc:
860 a.a.
41 a.a.*
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 3 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     calcium ion binding     2 terms  

 

 
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.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21352602 R.J.Konrad, J.S.Troutt, and G.Cao (2011).
Effects of currently prescribed LDL-C-lowering drugs on PCSK9 and implications for the next generation of LDL-C-lowering agents.
  Lipids Health Dis, 10, 38.  
19738285 J.S.Troutt, W.E.Alborn, G.Cao, and R.J.Konrad (2010).
Fenofibrate treatment increases human serum proprotein convertase subtilisin kexin type 9 levels.
  J Lipid Res, 51, 345-351.  
20227438 P.Costet (2010).
Molecular pathways and agents for lowering LDL-cholesterol in addition to statins.
  Pharmacol Ther, 126, 263-278.  
19687008 H.Li, B.Dong, S.W.Park, H.S.Lee, W.Chen, and J.Liu (2009).
Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.
  J Biol Chem, 284, 28885-28895.  
19443683 J.C.Chan, D.E.Piper, Q.Cao, D.Liu, C.King, W.Wang, J.Tang, Q.Liu, J.Higbee, Z.Xia, Y.Di, S.Shetterly, Z.Arimura, H.Salomonis, W.G.Romanow, S.T.Thibault, R.Zhang, P.Cao, X.P.Yang, T.Yu, M.Lu, M.W.Retter, G.Kwon, K.Henne, O.Pan, M.M.Tsai, B.Fuchslocher, E.Yang, L.Zhou, K.J.Lee, M.Daris, J.Sheng, Y.Wang, W.D.Shen, W.C.Yeh, M.Emery, N.P.Walker, B.Shan, M.Schwarz, and S.M.Jackson (2009).
A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.
  Proc Natl Acad Sci U S A, 106, 9820-9825.
PDB code: 3h42
20041144 J.R.Krycer, I.Kristiana, and A.J.Brown (2009).
Cholesterol homeostasis in two commonly used human prostate cancer cell-lines, LNCaP and PC-3.
  PLoS One, 4, e8496.  
19635789 S.Poirier, G.Mayer, V.Poupon, P.S.McPherson, R.Desjardins, K.Ly, M.C.Asselin, R.Day, F.J.Duclos, M.Witmer, R.Parker, A.Prat, and N.G.Seidah (2009).
Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route.
  J Biol Chem, 284, 28856-28864.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.