Mechanism of XIAP-Mediated Inhibition of Caspase-9

Abstract

The inhibitor of apoptosis (IAP) proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 A resolution. The structure reveals that the BIR3 domain forms a heterodimer with a caspase-9 monomer. Strikingly, the surface of caspase-9 that interacts with BIR3 also mediates its homodimerization. We demonstrate that monomeric caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases.

Caspase substrates

“…[23][24][25][26][27] In contrast to the many structures solved at the atomic level with synthetic substrate analogs bound to caspases, the only structure of a natural substrate bound is that of the viral caspase inhibitor p35 to caspase-8, which is trapped as a covalent adduct. 28 Interestingly, p35 contains a loop that interacts with a face of caspase-8 distant from the active site, possibly revealing an exosite interaction.…”

Design, Synthesis, and Characterization of a Potent, Nonpeptide, Cell-Permeable, Bivalent Smac Mimetic That Concurrently Targets Both the BIR2 and BIR3 Domains in XIAP

“…When compound 4 was added to uniformly 15 N labeled BIR2-BIR3 protein, 15 N HSQC spectra showed that many residues in the protein are affected by the compound (Supporting Information). Line width analysis of the residues within the core structural domains indicated that XIAP BIR2-BIR3 protein, with or without compound 4, has approximately the same molecular size, confirming that compound 4 does not cause dimerization of the protein, consistent with our gel filtration results.…”

“…15 N HSQC NMR spectra were recorded on a Bruker AVANCE 500MHz NMR spectrometer with samples containing 100 μM of the 15 N labeled proteins in 50 mM Tris (pH 7.2), 50 μM ZnCl 2 , 1 mM DTT at 25°C with or without test compound at a final concentration between 10-150 μM. The spectra were then compared in order to identify residues affected by the interaction with the test compound.…”

XLX is an IAP family member regulated by phosphorylation during meiosis

“…Biochemical analyses have led to the identification of the residues in the BIRs of cIAP-1, cIAP-2, and XIAP responsible for caspase binding. [4][5][6] The absence of these residues in ML-IAP and ILP-2 can explain the poor in vitro caspase-inhibition activity of these IAPs. 7,8 It has therefore been hypothesized that some IAPs may act instead as protein sinks to bind proapoptotic proteins, such as IAP antagonists.…”