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PDBsum entry 2w2q
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Hydrolase/receptor
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PDB id
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2w2q
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References listed in PDB file
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Key reference
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Title
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Structural and biochemical characterization of the wild type pcsk9-Egf(ab) complex and natural familial hypercholesterolemia mutants.
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Authors
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M.J.Bottomley,
A.Cirillo,
L.Orsatti,
L.Ruggeri,
T.S.Fisher,
J.C.Santoro,
R.T.Cummings,
R.M.Cubbon,
P.Lo surdo,
A.Calzetta,
A.Noto,
J.Baysarowich,
M.Mattu,
F.Talamo,
R.De francesco,
C.P.Sparrow,
A.Sitlani,
A.Carfí.
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Ref.
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J Biol Chem, 2009,
284,
1313-1323.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 1.
TR-FRET analyses of the PCSK9-LDLR interaction and its
inhibition. A, in a TR-FRET assay at neutral pH, EGF(A) and
EGF(AB) compete with the LDLR ectodomain for binding to PCSK9
with low/submicromolar IC[50] values. B, in the TR-FRET assay,
full-length WT PCSK9 and WT PCSK9ΔC are equipotent at
disrupting the interaction of labeled WT PCSK9 with labeled
LDLR, demonstrating that the C-terminal domain of PCSK9 is not
required for binding. In contrast with WT PCSK9ΔC, an
unprocessed form of PCSK9ΔC (S386A) was unable to disrupt the
interaction.
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Figure 3.
Crystal structure of the PCSK9ΔC-EGF(AB) complex. Ribbon (A)
and surface (B) representations, in the same orientation, of the
complex formed between PCSK9ΔC and EGF(A) (yellow). EGF(A)
contacts PCSK9 via the catalytic domain (red) but not the
prodomain (blue). The P′ helix, which is part of the catalytic
domain, is shown in pink. Dotted lines represent PCSK9 residues
for which no electron density was observed. C, transparent
surface representation of PCSK9ΔC; blue-labeled patches
indicate residues contacted by EGF(A) (yellow ribbon; the yellow
sphere is the Ca^2+ ion). Contacts are all less than 4 Å.
Asp-374 (PCSK9) is at the tip of a β-hairpin loop. EGF(A)
His-306 adopts a conformation that does not allow interaction
with Asp-374 at neutral pH. Ser-153 is the N terminus of the
PCSK9 catalytic domain generated upon autocleavage. Hydrogen
bonds are established between side chain atoms of the following
pairs of residues from PCSK9 and EGF(A): Arg-194 to Asp-310 and
Asn-295, Asp-238 to Asn-295, Thr-377 to Asn-309 and Asp-310, and
between backbone atoms for Thr-377 to Asp-310 and Phe-379 to
Cys-308. D, transparent surface representation of EGF(A);
yellow-labeled patches indicate residues contacted by PCSK9ΔC.
The protein is oriented as if removing PCSK9ΔC from the complex
shown in panel C followed by a 180° y axis rotation.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
1313-1323)
copyright 2009.
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