W323S Variant of Xiap-Bir3 Binds to SMAC but not Caspase-9

Abstract

The ability of the wild-type XIAP BIR3 domain as well as its Trp323Ser variant in inhibition of human caspase-9, binding to AVPFVASLPN (SMAC-peptide), SMAC protein, and mature caspase-9 was investigated. In order to investigate the role of W323 on these interactions, this residue was mutated to Serine. Circular dichroism as well as thermal denaturation studies showed that W323S mutation did not hamper proper folding of the protein. The dissociation constants for the interaction of the wild type BIR3 as well as its mutant to Smac-type peptide were found to be 1.8 and 27 muM, respectively. The inhibition of and binding to caspase-9 by wild-type BIR3 and its mutant were also compared. While the wild-type protein potently inhibited the enzyme, the mutant failed to do so. The lack of caspase-9 inhibition was due to absence of interaction of the mutant BIR3 with mature caspase-9. These results indicate that Trp323 of BIR3 plays a pivotal role both in maintaining necessary conformation for caspase-9 interaction and to a lesser extent, recognition of Smac-type peptide. Moreover, decreased stability of the mutant compared with the wild type indicates that W323 is essential for maintaining the stability BIR3-Smac-peptide complex.

A nonreplicating adenoviral vector that contains the wild‐type p53 transgene combined with chemotherapy for primary breast cancer

“…This was compared to the amount of inhibition observed in a gel‐based assay where the peptide concentration was fixed at 66.7 μ M , a concentration in excess of the best IC 50 app observed (Table I). The full BIR3 domain, with a reported K i of 10–20 n M 19, 21, 53 is an effective inhibitor in both the fluorescence assay, which uses a small fluorogenic peptide substrate mimic and in the gel‐based assay, which uses a natural substrate. In both activity assay formats, we observed moderate inhibition by many of the peptides.…”

“…In addition, the N‐terminus of the caspase‐9 small subunit binds to an exosite on the BIR3 domain. Critical interactions within these two regions of the BIR3 domain have been highlighted as essential for BIR3's interactions with caspase‐9 (Figure 1B) and its inhibitory properties 12, 19, 20. Many of these interactions are hydrogen bonds and salt bridges between the α5 helix of BIR3 and caspase‐9, often BIR3 side chains interacting with caspase‐9 backbone atoms.…”

A Mechanistic Insight into SMAC Peptide Interference with XIAP-Bir2 Inhibition of Executioner Caspases

“…[18][19][20] Thus, in order to address this issue, we synthesized a SMAC-type peptide that has been previously shown to relieve caspase-9 inhibition by XIAP. 21,22 This peptide was capable of antagonizing XIAP-Bir2 inhibition of caspase-3 and -7 in a dose-dependent manner ( Fig. 1; results are shown only for caspase-7).…”