PDBsum entry 2w2p

Hydrolase/receptor

PDB id: 2w2p

Contents

Protein chains

274 a.a. *

48 a.a. *

93 a.a. *

Metals

_CA

Waters ×31

* Residue conservation analysis

PDB id:

2w2p

Name:

Hydrolase/receptor

Title:

Pcsk9-deltac d374a mutant bound to wt egf-a of ldlr

Structure:

Proprotein convertase subtilisin/kexin type 9. Chain: a. Fragment: catalytic domain, residues 153-451. Synonym: proprotein convertase pc9, subtilisin/kexin-like protease pc9, neural apoptosis-regulated convertase 1, narc-1, pcsk9. Engineered: yes. Mutation: yes. Low-density lipoprotein receptor. Chain: e.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.62Å R-factor: 0.228 R-free: 0.270

Authors:

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.Carfi

Key ref:

M.J.Bottomley et al. (2009). Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants. J Biol Chem, 284, 1313-1323. PubMed id: 19001363 DOI: 10.1074/jbc.M808363200

Date:

03-Nov-08 Release date: 18-Nov-08

Protein chain

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

Seq: 692 a.a.

Struc: 274 a.a.*

Protein chain

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

Seq: 860 a.a.

Struc: 48 a.a.*

Protein chain

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

Seq: 692 a.a.

Struc: 93 a.a.

* PDB and UniProt seqs differ at 9 residue positions (black crosses)

Enzyme reactions

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

DOI no: 10.1074/jbc.M808363200

J Biol Chem 284:1313-1323 (2009)

PubMed id: 19001363

Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants.

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.

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 1313-1323) copyright 2009.

Figures were selected by an automated process.