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.

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

“…Nanobodies can be used to assist protein crystallization essentially in the same way. Examples of the protein structures solved with the help of nanobodies include several membrane receptors and transporters, proteins of bacterial secretion systems, and others (13)(14)(15)(16)(17)(18)(19)(20).…”

“…Therefore complete IgG antibodies (150 KDa) and F ab fragments (50 KDa) are not well suited as tools for protein NMR. In contrast, nanobodies (12)(13)(14)(15) can be successfully applied to studies of protein conformation and dynamics in solution by NMR.…”

“…Nanobodies can be used as NMR invisible probes, while the target proteins are isotopically labeled to record two-dimen-sional 1 H, 15 N or 1 H, 13 C chemical shift correlation spectra. Alternatively, isotopically 15 N-and/or 13 C-labeled nanobodies can be easily produced by standard isotope incorporation methods for proteins expressed in E. coli cells (69).…”

Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies

“…Raising and Producing Llama V HH against C-DCX-Antibody Fab fragments are established as crystallization helpers (17,18). The corresponding fragments of camelid heavy chainonly antibodies (V HH or Nanobodies TM ) have only one "Ig domain" (19,20).…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…Ab peptides arise from a proteolytic cleavage of the transmembrane glycoprotein amyloid precursor protein (APP) following the action of two secretases [140]. First, the b-secretase generates the APPsb fragment (secreted in the plasma) and the C99 fragment (still membrane anchored) [141]. This latter is then cleaved by the gsecretase into a variety of peptides including the amyloidogenic Ab 40 and Ab 42 fragments.…”