Pearlstein, Robert, Hu, Qi-Ying, Zhou, Jing, Yowe, David and Levell, Julian (2010) NEW HYPOTHESES ABOUT THE STRUCTURE-FUNCTION OF PCSK9, PART 1: ANALYSIS OF THE EGF-A DOCKING SITE USING WATERMAP. PROTEINS: Structure, Function, and Bioinformatics.

Abstract

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to and targets the low density lipoprotein receptor (LDL-R) for lysozomal degradation. Down regulation of LDL-R leads to dyslipidemia and cardiovascular disease, whereas the beneficial effects of PCSK9 mediated regulation of LDL-R are currently unknown. We used Biacore to measure binding between PCSK9 and LDL-R, and between PCSK9 and the epidermal growth factor-like repeat A (EGF-A) of LDL-R. EGF-A exhibits fast kon and fast koff, whereas receptor binding and unbinding are slower. We also measured binding between PCSK9 and EGF-A using isothermal titration calorimetry (ITC). Heat transfer was not detected, consistent with an entropically driven process. We analyzed and compared the crystal structures of wild type vs. mutant (Asp374Tyr) PCSK9 bound to EGF-A using a new capability called WaterMap, which predicts the energies and positions of protein hydration sites. EGF-A was found to rely heavily on stable hydration sites for binding, placing polar atoms at these positions. Entropic gain, and fast kon are expected under these conditions. Insufficient availability of unstable hydration sites needed to slow EGF-A unbinding explains the relatively fast koff observed. The considerably slower koff observed for PCSK9-LDL-R unbinding is consistent with the existence of additional unstable hydration sites at a second docking site outside of EGF-A. WaterMap further suggests that the slower koff observed for Asp374Tyr PCSK9 is due to a net gain of unstable hydration sites resulting from this mutation. We hypothesize that endosomal pH speeds up kon for both LDL-R and EGF-A due to a gain of additional stable hydration sites at or near the EGF-A interface, and slows koff for LDL-R due to gain of additional unstable hydration sites.

Item Type:	Article
Additional Information:	The draft has been reviewed by patent department (Paul Paglierani) prior to the OAK system.
Keywords:	Pcsk9, protein protein interaction, EGF-A, watermap
Date Deposited:	13 Oct 2015 13:17
Last Modified:	13 Oct 2015 13:17
URI:	https://oak.novartis.com/id/eprint/1248