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J. Biol. Chem., Vol. 284, Issue 2, 1313-1323, January 9, 2009

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

Matthew J. Bottomley¹, Agostino Cirillo, Laura Orsatti, Lionello Ruggeri, Timothy S. Fisher, Joseph C. Santoro, Richard T. Cummings, Rose M. Cubbon, Paola Lo Surdo, Alessandra Calzetta, Alessia Noto, Jennifer Baysarowich, Marco Mattu, Fabio Talamo, Raffaele De Francesco², Carl P. Sparrow, Ayesha Sitlani, and Andrea Carfí³

From the Department of Biochemistry, Istituto di Ricerca di Biologia Molecolare "P. Angeletti", Via Pontina Km 30.600, 00040 Pomezia (Rome), Italy and the Division of Cardiovascular Diseases, Merck Research Laboratories, Rahway, New Jersey 07065

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 PCSK9C-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 PCSK9C 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 PCSK9C 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.

Received for publication, November 3, 2008 , and in revised form, November 7, 2008.

The atomic coordinates and structure factors (codes 2W2M, 2W2N, 2W2O, 2W2P, and 2W2Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

² Present address: Istituto Nazionale Genetica Molecolare, via Francesco Sforza 28, 20122 Milano, Italy.

¹ To whom correspondence may be addressed. Tel.: 39-06-91093-502; Fax: 39-06-91093-225; E-mail: matthew_bottomley{at}merck.com.

³ To whom correspondence may be addressed. Tel.: 39-06-91093-550; Fax: 39-06-91093-625; E-mail: andrea_carfi{at}merck.com.

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