An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.

These structures span all three caspase subgroups, and provide a basis for inferring substrate and inhibitor binding, as well as selectivity for the entire caspase family. This information will influence the design of selective caspase inhibitors to further elucidate the role of caspases in biology and hopefully lead to the design of therapeutic agents to treat caspase-mediated diseases, such as rheumatoid arthritis, certain neurogenerative diseases and stroke.

Interleukin-1␤-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1␤ (pre-IL-1␤) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P 1 aspartate residue. We have examined the kinetics of processing pre-IL-1␤ to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1␤, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADPribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 ؋ 10 ICE 1 is the prototypical member of a new family of mammalian cysteine proteases (the caspases) 2 that is distinct from cysteine proteases in the papain superfamily (1-3). The mutagenesis experiments and crystal structure reported by Wilson et al. (4) revealed a different active site geometry and catalytic mechanism for ICE than observed for papain. The structure of the ICE active site contains a Cys-His catalytic diad, and two Arg residues that confer high selectivity for peptidyl substrates with Asp residues at the P 1 position (Nterminal to the scissile bond) (4, 5). Although ICE has recently been reported to cleave other proteins in vitro (6, 7), it was identified from its essential role in processing the inactive 31-kDa precursor of interleukin-1␤ (pre-IL-1␤) to the mature 17-kDa cytokine (8).In 1993, Yuan et al. (9) reported the sequence of the Caenorhabditis elegans programmed cell death gene ced-3. This gene is 29% identical to human ICE. Due to the central role of the CED-3 protein in C. elegans apoptosis, Yuan and colleagues deduced that ICE or ICE homologs might play a similar role in mammalian apoptosis. Overexpression of ICE in rat fibroblast, mammalian COS cells, and neuronal cell lines demonstrated that this protease can indeed induce apoptosis (6, 10, 11). Subsequently, Kuida et al. (12) confirmed an in vivo role for ICE in Fas-mediated apoptosis by disruption of the murine ICE gene.A family of ICE-related proteases (the caspases) was discovered by searching human cDNA libraries for sequences homologous to 14). At present, at least 10 human homologs of ICE possessing cysteine protease activity have been identified. These homologs can be grouped by sequence similarity into three subfamilies. The most closely related homologs to ICE are TX (caspase-4, also denoted ICH-2 or ICErel II) (15-17) and TY (caspase-5, also denoted ICE rel III) (17, 18), which are 58 and 57% identical to ICE at the amino acid level. Another homolog, ICH-1 (caspase-2, corresponding to the murine Nedd-2 gene) (19,20), is 20% identical to ICE and belongs to a distinct subfamily. A third gr...

Interactions mediated by phosphoryl-Thr183 induce structural changes that direct the domains and active-site residues of P38gamma into a conformation consistent with catalytic activity. The conformation of the phosphorylation loop is likely to be similar in all activated MAP kinases, but not all activated MAP kinases form dimers.

This is the first JNK structure to be determined, providing a unique opportunity to compare structures from the three MAP kinase subfamilies. The structure reveals atomic-level details of the shape of JNK3 and the interactions between the kinase and the nucleotide. The misalignment of catalytic residues and occlusion of the active site by the phosphorylation lip may account for the low activity of unphosphorylated JNK3. The structure provides a framework for understanding the substrate specificity of different JNK isoforms, and should aid the design of selective JNK3 inhibitors.