title: Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants.
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.
authors.affiliation: Department of Biochemistry, Istituto di Ricerca di Biologia Molecolare "P. Angeletti", Via Pontina Km 30.600, 00040 Pomezia (Rome), Italy. matthew_bottomley@merck.com
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Bottomley
authors.forename: Agostino
authors.initials: A
authors.lastname: Cirillo
authors.forename: Laura
authors.initials: L
authors.lastname: Orsatti
authors.forename: Lionello
authors.initials: L
authors.lastname: Ruggeri
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Fisher
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Santoro
authors.forename: Richard T
authors.initials: RT
authors.lastname: Cummings
authors.forename: Rose M
authors.initials: RM
authors.lastname: Cubbon
authors.forename: Paola
authors.initials: P
authors.lastname: Lo Surdo
authors.forename: Alessandra
authors.initials: A
authors.lastname: Calzetta
authors.forename: Alessia
authors.initials: A
authors.lastname: Noto
authors.forename: Jennifer
authors.initials: J
authors.lastname: Baysarowich
authors.forename: Marco
authors.initials: M
authors.lastname: Mattu
authors.forename: Fabio
authors.initials: F
authors.lastname: Talamo
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
authors.forename: Carl P
authors.initials: CP
authors.lastname: Sparrow
authors.forename: Ayesha
authors.initials: A
authors.lastname: Sitlani
authors.forename: Andrea
authors.initials: A
authors.lastname: Carfí
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D018360:Crystallography, X-Ray; D006801:Humans; D006938:Hyperlipoproteinemia Type II; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases