The inhibitor-of-apoptosis (IAP) family of proteins, originally identified in baculoviruses, regulate programmed cell death in a variety of organisms. IAPs inhibit specific enzymes (caspases) in the death cascade and contain one to three modules of a common 70-amino-acid motif called the BIR domain. Here we describe the nuclear magnetic resonance structure of a region encompassing the second BIR domain (BIR2) of a human IAP family member, XIAP (also called hILP or MIHA). The structure of the BIR domain consists of a three-stranded antiparallel beta-sheet and four alpha-helices and resembles a classical zinc finger. Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were found to be critical for inhibiting caspase-3. The absence or presence of these residues may explain the differences in caspase inhibition observed for different truncated and full-length IAPs. Our data further indicate that these residues may bind to the active site and that the BIR domain may interact with an adjacent site on the enzyme.

The severe acute respiratory syndrome (SARS) virus belongs to the Coronaviridea family of viruses. Its virion encodes several proteins including a replicase and four structural proteins. Here we describe the three-dimensional structure of the N-terminal domain of the SARS coronavirus (CoV) nucleocapsid protein. The protein consists of a five-stranded beta sheet with a folding topology distinct from other RNA-binding proteins. Single-stranded RNAs bind to the protein surface at the junction between a flexible, positively charged beta hairpin and the core structure. NMR-based screening was used to identify low molecular weight compounds that bind to this site.

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 threedimensional structure of the Bir3 domain of XIAP, determined by NMR spectroscopy, resembles a classical zinc finger and consists of five ␣-helices, a threestranded ␤-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.Programmed cell death is a tightly regulated process that is critical for normal development and tissue homeostasis and when dysregulated can lead to a variety of diseases such as neurodegenerative disorders and cancer (1). One class of proteins that negatively regulates cell death signaling is the inhibitor of apoptosis proteins (IAPs).1 IAPs are highly conserved and have been found in many species (2-6). The members of this family are characterized by having one or more baculovirus IAP repeats called Bir domains. Bir domains consist of approximately 70 amino acids that contain the characteristic signature sequence CX 2 CX 16 HX 6 C (7, 8). Some IAPs also contain a C-terminal ring finger that contains one zinc atom chelated to three cysteines and one histidine and another zinc ligated to four cysteines (9).One of the major functions of the IAPs is their ability to bind to and inhibit the cysteine proteases known as caspases (10, 11), which play a key role in the execution of programmed cell death (12). Caspase inhibition by the IAPs can directly explain their antiapoptotic activities. For human XIAP, the region responsible for inhibiting caspases-3 and -7 was localized to a fragment containing the second Bir domain (13). Although the Bir domain was necessary for caspase-3 inhibition, residues outside of the Bir2 domain were also found to be critical for inhibiting caspase-3 (14). On the basis of site-directed mutagenesis and NMR studies on the interaction of XIAP with caspase-3, it was postulated that the residues N-terminal to the Bir2 domain of XIAP bind to the active site of this enzyme (14).XIAP also inhibits caspase-9. However, a different portion of XIAP is involved. Recently, Deveraux et al. (15) have shown that it is the Bir3 and ring finger of XIAP that potently inhibits caspase-9. Since neither the ring finger nor the Bir3 domain of XIAP was suf...