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Journal of Biochemistry Advance Access originally published online on December 19, 2006
Journal of Biochemistry 2007 141(3):293-299; doi:10.1093/jb/mvm028
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© 2006 The Japanese Biochemical Society.

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

Jamshid Davoodi1,*, Azadeh Mohammad-Gholi1, Ali Es-haghi1 and Alex MacKenzie2

1Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; and 2Apoptosis Research Center, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada

*To whom correspondence should be addressed at: Jamshid Davoodi, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. Tel: +98-21-6696-9190, Fax: +98-21-6640-4680, E-mail: jdavoodi{at}ibb.ut.ac.ir

Received November 4, 2006; Accepted December 11, 2006


  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 µM, 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.

Key Words: caspase-9 inhibition, Smac, XIAP BIR3

Abbreviations: BIR, baculoviral IAP repeat; NAIP, neuronal apoptosis inhibitory protein; XIAP, X-linked inhibitor of apoptosis protein


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