Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

ABSTRACTcDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show -40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share -40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and m9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-LeuSer-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.Factor V is a large and asymmetric glycoprotein that circulates in plasma and is an essential component of the blood coagulation cascade (1, 2). During coagulation, the procofactor factor V is converted to the active cofactor, factor Va, via limited proteolysis by a-thrombin (3-5). Factor Va is a cofactor for the seine protease factor Xa, and together, factors Va and Xa assemble on a cellular or phospholipid surface with divalent metal ions to form the prothrombinase complex (1, 6-11). This complex enhances factor Xa activity -350,000 fold. The prothrombinase complex is analogous to another complex that proteolytically cleaves zymogen factor X to active enzyme factor Xa-this other "ten-ase" complex is composed of a serine protease (factor IXa), a cofactor (factor VIIMa), phospholipid, and calcium (13, 14).In addition to the similarities between serine proteases (factors Xa and IXa) and in overall enzyme complex architecture, the cofactors (factor Va and factor VIIIa) are very similar proteins structurally and functionally (13-16). Heavy and light chains of bovine factor Va and porcine factor VIIIa possess amino acid-sequence homology at the amino-terminal portion of the chains-regions of homology that are also homologous to regions in the triplicated domain structure of ceruloplasmin, the primary transport protein for copper in plasma. Available data therefore suggest that factor V, factor VIII, and ceruloplasmin are members of a family of structurally related proteins (15).The molecular cloning and sequencing of human factor VIII and human ceruloplasmin gives evidence for a common domain structure and has enabled detailed comparison of their structures (16,(17)(18)(19)(20). Recently Kane and Davie (21) published a partial cDNA sequence for human factor V that coded for "40% of the molecule. This cDNA coded for the light chain and a small ...

We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin‐1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD‐2/ICH‐1L and CED‐3. A computer homology model of TX was constructed based on the X‐ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL‐1 beta from pro‐IL‐1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced‐3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.

The immunophilin FKBP12 is one of the most abundant and conserved proteins in biology. It is the primary receptor for the immunosuppressant actions of the drug FK506 in whose presence FKBP12 binds to and inhibits calcineurin, disrupting interleukin formation in lymphocytes. The physiologic functions of FKBP12 are less clear, although the protein has been demonstrated to physiologically interact with the inositol 1,4,5-trisphosphate receptor (IP 3 R), the ryanodine receptor, and the type 1 transforming growth factor ␤ receptor. We now report that FKBP12 binds the IP 3 R at residues 1400 -1401, a leucyl-prolyl dipeptide epitope that structurally resembles FK506. We further demonstrate that binding to IP 3 R at this site enables FKBP12 to interact with calcineurin, presumably to anchor the phosphatase to IP 3 R and modulate the receptor's phosphorylation status. We propose that FK506 promotes an FKBP12-calcineurin interaction by mimicking structurally similar dipeptide epitopes present within proteins that use FKBP12 to anchor calcineurin to the appropriate physiologic substrates.The immunophilins are proteins that bind the immunosuppressant drugs cyclosporin A (CsA), 1 FK506, and rapamycin with high affinity and are responsible for their therapeutic actions (for review, see Refs. 1 and 2). Cyclosporin A, a cyclic undecapeptide, binds to members of the cyclophilin family, whereas the structurally unrelated FK506 and rapamycin bind to the family of FK506 binding proteins (FKBPs). Although the cyclophilins and FKBPs lack amino acid sequence homology, both classes of proteins display peptidyl-prolyl isomerase activity, which is inhibited by their respective immunosuppressant ligands. However, inhibition of this rotamase activity does not explain immunosuppression, as some potent ligands of the immunophilins inhibit rotamase activity but lack immunosuppressant effects (3). Immunosuppression appears to stem from the binding of the drug-immunophilin complex to the calciumactivated phosphatase calcineurin (CN) to inhibit catalytic activity resulting in an accumulation of phosphorylated CN substrates (4). One of these substrates, the transcription factor NFAT (nuclear factor of activated T-cells) in its unphosphorylated state passes from the cytoplasm to the nucleus to stimulate interleukin-2 formation. Following treatment with immunosuppressant drugs, phosphorylated levels of NFAT accumulate in the cytoplasm and are unable to enter the nucleus with the associated decrease in interleukin-2 formation being involved in immunosuppressant actions (5, 6).Whereas pharmacologic actions of immunosuppressant drugs are readily explained by the above model, the physiologic roles of the immunophilins remain obscure despite the fact that they are among the most abundant and conserved proteins in biology. A few proteins, such as collagen and transferrin, have been shown to serve as substrates for immunophilin rotamase activity (7,8). However, it is unclear whether these are the sole or principal physiologic substrates for the rotamase a...

Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-Å resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-Å resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 M). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K m increased Ϸ5-fold and the V max decreased Ϸ30%. In contrast, the topoisomerase IV ATP K m decreased by a factor of 6, and the V max increased Ϸ2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.Type II topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. Bacterial genomes encode two type II topoisomerases, DNA gyrase and topoisomerase IV (TopoIV), that function in DNA replication. DNA gyrase is unique in coupling the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. In the absence of the ATP substrate, DNA gyrase can relax negatively supercoiled plasmid DNA. These activities result from the enzyme's ability to wrap (Ϸ150 bp) DNA (23, 31) around itself upon binding the DNA substrate. This DNA wrapping preferentially presents the T-segment (transported DNA segment) to the gyrase-DNA complex so that the introduction of negative supercoils is the primary outcome. In contrast, TopoIV and other eukaryotic type II topoisomerases only bind a Ϸ30-bp region of DNA (20,35). TopoIV utilizes the energy of ATP hydrolysis to decatenate newly replicated chromosomal DNA but also has the ability to relax positive and negative DNA supercoils in an ATP-dependent manner (8,43).In prokaryotes, these type II topoisomerases are composed of two subunits. In Escherichia coli, the gyrase subunits are named A and B and the corresponding TopoIV subunits are named C and E. For each enzyme, these subunits combine into a heterotetrameric (gyrase, A 2 B 2 ; and TopoIV, C 2 E 2 ) complex to form the functional enzymes. I...