NMR Structure and Mutagenesis of the Third Bir Domain of the Inhibitor of Apoptosis Protein XIAP

doi:10.1074/jbc.M006226200

NMR Structure and Mutagenesis of the Third Bir Domain of the Inhibitor of Apoptosis Protein XIAP*

From the ^‡Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, Illinois 60064 and the ^§Biochemistry Department, Idun Pharmaceutical Inc., La Jolla, California 92037

Abstract

The inhibitor of apoptosis proteins (IAPs) regulate the caspase family of cysteine proteases, which play an important role in the execution of programmed cell death. Human X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases-3, -7, and -9. Here we show that the Bir3 domain is the minimal region of XIAP that is needed for potent caspase-9 inhibition. The three-dimensional structure of the Bir3 domain of XIAP, determined by NMR spectroscopy, resembles a classical zinc finger and consists of five α-helices, a three-stranded β-sheet, and a zinc atom chelated to three cysteines and one histidine. The structure of the Bir3 domain is similar to that of the Bir2 domain of XIAP but differs from the previously determined structure of the Bir3 domain of MIHB. Based on site-directed mutagenesis, we have identified the regions of the Bir3 domain of XIAP that are important for inhibiting caspase-9. Despite the structural similarities of the Bir2 and Bir3 domain of XIAP, a different set of residues were found to be critical for inhibiting the individual caspases. These results suggest that XIAP inhibits caspase-3 and caspase-9 in a different manner.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
↵¶ To whom correspondence should be addressed: Abbott Laboratories, 100 Abbott Park Rd., D-47G, AP10-LL, Abbott Park, IL 60064-6098. Tel.: 847-937-1201; Fax: 847-938-2478; E-mail: stephen.fesik@abbott.com.
Published, JBC Papers in Press, August 8, 2000, DOI 10.1074/jbc.M006226200
↵2 J. S. Krebs and J. C. Wu, unpublished data.
Abbreviations:

IAP

inhibitor of apoptosis protein

XIAP

X-linked inhibitor of apoptosis protein

Bir

baculovirus IAP repeat

NOE

nuclear Overhauser effect

Chaps

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

bis-Tris

bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane

HSQC

heteronuclear single quantum correlation
- Received July 13, 2000.
- Revision received August 7, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.