BacMam production and crystal structure of nonglycosylated apo human furin at 1.89 Å resolution

Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium‐dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis...

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Bibliographic Details
Published in:Acta crystallographica. Section F, Structural biology communications Structural biology communications, 2019-04, Vol.75 (4), p.239-245
Main Authors: Pearce, Kenneth H., Overton, Laurie K., Gampe, Robert T., Barrett, George B., Taylor, J. David, McKee, David D., Campobasso, Nino, Nolte, Robert T., Reid, Robert A.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
apo
Apoproteins - chemistry
BacMam
Biochemistry - methods
Calcium
Cancer
CHO Cells
Cricetinae
Cricetulus
Crystal structure
Crystallography
Crystallography, X-Ray
Crystals
Fibrosis
Furin
Furin - chemistry
Glycosylation
HEK293 Cells
Homeostasis
Humans
hydrolase
Kexin
nonglycosylated
Proprotein convertases
Protein Domains
Protein purification
Protein Structure, Secondary
Proteins
Proteolysis
Research Communications
Serine
serine proteinase
Site-directed mutagenesis
Subtilisin
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Summary:Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium‐dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site‐directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure‐guided drug‐discovery campaign. The production of nonglycosylated human furin ectodomain via the BacMam expression system is reported. The protein is equally active compared with the glycosylated protein and is readily crystallized; X‐ray diffraction was used to solve a high‐resolution structure.
ISSN:2053-230X
2053-230X
DOI:10.1107/S2053230X19001419