Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation 
of the hepatic low-density lipoprotein receptor (LDL-R) and is therefore a 
prominent therapeutic target for reducing LDL-cholesterol. The C-terminal domain 
of PCSK9 is unlikely to be involved in a direct extracellular interaction with 
the LDL-R. We probed the importance of the C-terminus for the degradation of the 
LDL-R by designing seven de novo mutants of PCSK9 that fill potential druggable 
cavities. The mutants were tested for their ability to diminish LDL uptake in 
human HepG2 cells and for affinity towards a calcium independent mutant of the 
EGF(A) domain of the human LDL-R. The later was done by a newly developed 
surface plasmon resonance-based assay format. We identified three mutant 
proteins (G517R, V610R and V644R) with decreased ability to block LDL uptake 
into HepG2 cells. These mutations define areas outside the direct interaction 
area between PCSK9 and the LDL-R that could be targeted to inhibit the PCSK9 
triggered degradation of the LDL-R. We also describe the mechanistic 
rationalisation of the affinity changes seen with the natural occurring human 
D374Y (gain of function) mutation causing severe hypercholesterolaemia. The 
action of this mutant is due to a significantly decreased dissociation rate 
constant, whereas the mutation does not affect the association rate constant.