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 PCSK9 Delta C-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 PCSK9 Delta C 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, LDLRH306Y 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 PCSK9 Delta C 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.
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.L. Surdo, A. Calzetta, A. Noto, J. Baysarowich, M. Mattu, F. Talamo, R. De Francesco, C.P. Sparrow, A. Sitlani, A. Carfí. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 284:2(2009), pp. 1313-1323.
|Titolo:||Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants|
|Parole Chiave:||Density-lipoprotein receptor; autosomal-dominant hypercholesterolemia; convertase subtilisin/kexin type-9; PCSK9 gene; secreted PCSK9; LDL receptors; molecular-basis; mutations; binding; domain|
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
Settore BIO/11 - Biologia Molecolare
Settore BIO/13 - Biologia Applicata
|Data di pubblicazione:||2009|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1074/jbc.M808363200|
|Appare nelle tipologie:||01 - Articolo su periodico|