A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles
Samantha K. Sarkar, Alexander C.Y. Foo, Angela Matyas, Tanja Kosenko, Natalie K. Goto, Ariela Vergara-Jaque, Thomas A. Lagace
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30-40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9’s prodomain. Here we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N-terminus adopted helical structure in a membrane-mimetic environment; this effect was greatly enhanced by an R46L substitution representing an athero-protective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested the transient α-helix aligns multiple polar residues to interact with positive-charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, F515L) inhibited LDL binding, which was abolished for the R496W variant. These studies inform on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, we report the initial identification of FH-associated mutations that diminish the ability of PCSK9 to bind LDL, supporting that LDL association in the circulation inhibits PCSK9 activity.
Circular dichroism, Secondary structure, Ligand binding, Vesicle interactions, Biochemistry