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PDBsum entry 3gcw
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Protein binding
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PDB id
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3gcw
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References listed in PDB file
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Key reference
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Title
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Antagonism of secreted pcsk9 increases low density lipoprotein receptor expression in hepg2 cells.
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Authors
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M.C.Mcnutt,
H.J.Kwon,
C.Chen,
J.R.Chen,
J.D.Horton,
T.A.Lagace.
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Ref.
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J Biol Chem, 2009,
284,
10561-10570.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
10561-10570)
copyright 2009.
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