Characterisation of de novo mutations in the C-terminal domain of proprotein convertase subtilisin/kexin type 9.
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.
Discovery Sciences, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden stefan.geschwindner@astrazeneca.com wolfgang.knecht@biol.lu.se.
Stefan
S
Geschwindner
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Gunilla M K
GM
Andersson
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Hans-Georg
HG
Beisel
Discovery Sciences, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Sebastian
S
Breuer
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Carina
C
Hallberg
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Britt-Marie
BM
Kihlberg
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Ann-Margret
AM
Lindqvist
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Gavin
G
O'Mahony
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Alleyn T
AT
Plowright
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Florian
F
Raubacher
CVMD Innovative Medicines, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden Present address: Department of Biology, Molecular Cell Biology & Lund Protein Production Platform, Lund University, 22362 Lund, Sweden stefan.geschwindner@astrazeneca.com wolfgang.knecht@biol.lu.se.
Wolfgang
W
Knecht
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D056945:Hep G2 Cells; D006801:Humans; D006937:Hypercholesterolemia / Q000235:genetics; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D020125:Mutation, Missense; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D011973:Receptors, LDL / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D008077:Lipoproteins, LDL; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
PCSK9; SPR; drug discovery; protein-engineering