title: Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.
abstract: The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.
authors.affiliation: pRED Pharma Research and Early Development, Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland.
authors.forename: David W
authors.initials: DW
authors.lastname: Banner
authors.forename: Bernard
authors.initials: B
authors.lastname: Gsell
authors.forename: Jörg
authors.initials: J
authors.lastname: Benz
authors.forename: Julian
authors.initials: J
authors.lastname: Bertschinger
authors.forename: Dominique
authors.initials: D
authors.lastname: Burger
authors.forename: Simon
authors.initials: S
authors.lastname: Brack
authors.forename: Simon
authors.initials: S
authors.lastname: Cuppuleri
authors.forename: Maja
authors.initials: M
authors.lastname: Debulpaep
authors.forename: Alain
authors.initials: A
authors.lastname: Gast
authors.forename: Dragan
authors.initials: D
authors.lastname: Grabulovski
authors.forename: Michael
authors.initials: M
authors.lastname: Hennig
authors.forename: Hans
authors.initials: H
authors.lastname: Hilpert
authors.forename: Walter
authors.initials: W
authors.lastname: Huber
authors.forename: Andreas
authors.initials: A
authors.lastname: Kuglstatter
authors.forename: Eric
authors.initials: E
authors.lastname: Kusznir
authors.forename: Toon
authors.initials: T
authors.lastname: Laeremans
authors.forename: Hugues
authors.initials: H
authors.lastname: Matile
authors.forename: Christian
authors.initials: C
authors.lastname: Miscenic
authors.forename: Arne C
authors.initials: AC
authors.lastname: Rufer
authors.forename: Daniel
authors.initials: D
authors.lastname: Schlatter
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.forename: Martine
authors.initials: M
authors.lastname: Stihle
authors.forename: Ralf
authors.initials: R
authors.lastname: Thoma
authors.forename: Martin
authors.initials: M
authors.lastname: Weber
authors.forename: Armin
authors.initials: A
authors.lastname: Ruf
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019540:Area Under Curve; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003460:Crystallization; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D020349:Surface Plasmon Resonance; D014961:X-Ray Diffraction
chemical_list: D007140:Immunoglobulin Fab Fragments; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human
keywords: antibodies; drug discovery and design; protein crystallization