The structure of XIAP BIR2: understanding the selectivity of the BIR domains

Abstract

XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain-caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3-caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2-caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2-tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.

Keywords: AVPI; BIR domains; SMAC; XIAP; apoptosis; caspases; extrinsic pathway; inhibitor of apoptosis; peptide complex.

Figure 1

Overlay of the XIAP BIR2 crystal structure at 1.35 Å resolution (orange), the NMR structure (green; PDB entry 1c9q; Sun et al., 1999 ▶) and the caspase-3-bound form (light blue; PDB entry 1i3o; Riedl et al., 2001 ▶). The construct used in crystallization removes the flexible and variable preceding linker region as well as residues from the C-terminus. For the apo structure, only chain A is shown; in the other molecule the N-­terminus is disordered up to residue 159. The zinc is shown as a sphere and Trp210, which creates the floor of the binding groove, is shown in stick representation.

Figure 2

The ATAA tetrapeptide bound to the XIAP BIR2 domain, showing the protein surface as represented in PyMOL. Important residues are labeled. Hydrogen bonds are shown as yellow dashes.

Figure 3

(a) Overlay of ATAA (green), SVPI (orange) and AIAV (blue). A combination of peptide movement and rotation of Gln197 allows the protein to accommodate the differently sized P4 residues. Gln197 was observed in two conformations in the SVPI structure. (b) Overlay of SVPI (orange) and AVPI (red). Instead of Gln197 moving away, the peptide is pushed out. (c) Overlay of SVPI (orange) and AMRV (blue). The arginine causes the backbone to shift enough that the hydrogen bond to the Leu207 backbone is lost.

Figure 4

(a) Overlay of AVPI structures: XIAP BIR2–SVPI in orange, XIAP BIR3–AVPI in cyan (PDB entry 1g73; Wu et al., 2000 ▶), MLIAP–AVPI in turquoise (PDB entry 1oxq; Franklin et al., 2003 ▶) and CIAP1 BIR3–AVPI in dark blue (PDB entry 3d9u; Kulathila et al., 2009 ▶). Note that Lys206 is a glycine in XIAP BIR3, MLIAP and CIAP BIR3 and this restricts the size of the pocket, so that P4 is buried less deeply. (b) Overlay of XIAP BIR2 with AIAV bound (green) and DIAP1 BIR1 with the Grim peptide bound (purple; AIAY from Grim shown; PDB entry 1seo). In this case, Lys208 is a glycine in DIAP1 BIR1, allowing the large tyrosine at P4 to fit.