Structural basis for the activation of human procaspase-7.

Caspases form a family of proteinases required for the initiation and execution phases of apoptosis. Distinct proapoptotic stimuli lead to activation of the initiator caspases-8 and -9, which in turn activate the common executioner caspases-3 and -7 by proteolytic cleavage. Whereas crystal structures of several active caspases have been reported, no three-dimensional structure of an uncleaved caspase zymogen is available so far. We have determined the 2.9-A crystal structure of recombinant human C285A procaspase-7 and have elucidated the activation mechanism of caspases. The overall fold of the homodimeric procaspase-7 resembles that of the active tetrameric caspase-7. Each monomer is organized in two structured subdomains connected by partially flexible linkers, which asymmetrically occupy and block the central cavity, a typical feature of active caspases. This blockage is incompatible with a functional substrate binding site/active site. After proteolytic cleavage within the flexible linkers, the newly formed chain termini leave the cavity and fold outward to form stable structures. These conformational changes are associated with the formation of an intact active-site cleft. Therefore, this mechanism represents a formerly unknown type of proteinase zymogen activation.

Dimer formation drives the activation of the cell death protease caspase 9.

A critical step in the induction of apoptosis is the activation of the apoptotic initiator caspase 9. We show that at its normal physiological concentration, caspase 9 is primarily an inactive monomer (zymogen), and that activity is associated with a dimeric species. At the high concentrations used for crystal formation, caspase 9 is dimeric, and the structure reveals two very different active-site conformations within each dimer. One site closely resembles the catalytically competent sites of other caspases, whereas in the second, expulsion of the "activation loop" disrupts the catalytic machinery. We propose that the inactive domain resembles monomeric caspase 9. Activation is induced by dimerization, with interactions at the dimer interface promoting reorientation of the activation loop. These observations support a model in which recruitment by Apaf-1 creates high local concentrations of caspase 9 to provide a pathway for dimer-induced activation.

Crystal structure of the anthrax lethal factor.

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.

Mechanism-based inactivation of caspases by the apoptotic suppressor p35.

Caspases play a crucial role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, one of which--p35--has so far only been found in baculoviruses. P35 has previously been shown to rapidly form essentially irreversible complexes with its target caspases in a process that is accompanied by peptide bond cleavage. To determine the protease-inhibitory pathway utilized by this very selective protease inhibitor, we have analyzed the thermodynamic and kinetic stability of the protein. We show that the conformation of p35 is stabilized following cleavage within its reactive site loop. An inactive catalytic mutant of caspase 3 is bound by p35, but much less avidly than the wild-type enzyme, indicating that the protease catalytic nucleophile is required for stable complex formation. The inhibited protease is trapped as a covalent adduct, most likely with its catalytic Cys esterified to the carbonyl carbon of the scissile peptide bond. Together these data reveal that p35 is a mechanism-based inactivator that has adopted an inhibitory device reminiscent of the widely distributed serpin family, despite a complete lack of sequence or structural homology.

Structural basis for the inhibition of caspase-3 by XIAP.

The molecular mechanism(s) that regulate apoptosis by caspase inhibition remain poorly understood. The main endogenous inhibitors are members of the IAP family and are exemplified by XIAP, which regulates the initiator caspase-9, and the executioner caspases-3 and -7. We report the crystal structure of the second BIR domain of XIAP (BIR2) in complex with caspase-3, at a resolution of 2.7 A, revealing the structural basis for inhibition. The inhibitor makes limited contacts through its BIR domain to the surface of the enzyme, and most contacts to caspase-3 originate from the N-terminal extension. This lies across the substrate binding cleft, but in reverse orientation compared to substrate binding. The mechanism of inhibition is due to a steric blockade prohibitive of substrate binding, and is distinct from the mechanism utilized by synthetic substrate analog inhibitors.

Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

Crystal structure of the apoptotic suppressor CrmA in its cleaved form.

BACKGROUND: Cowpox virus expresses the serpin CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic responses of infected host cells. The targets of CrmA are members of the caspase family of proteases that either initiate the extrinsic pathway of apoptosis (caspases 8 and 10) or trigger activation of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 (caspase 1). RESULTS: We have determined the structure of a cleaved form of CrmA to 2.26 A resolution. CrmA has the typical fold of a cleaved serpin, even though it lacks the N-terminal half of the A helix, the entire D helix, and a portion of the E helix that are present in all other known serpins. The reactive-site loop of CrmA was mutated to contain the optimal substrate recognition sequence for caspase 3; however, the mutation only marginally increased the ability of CrmA to inhibit caspase 3. Superposition of the reactive-site loop of alpha1-proteinase inhibitor on the cleaved CrmA structure provides a model for virgin CrmA that can be docked to caspase 1, but not to caspase 3. CONCLUSIONS: CrmA exemplifies viral economy, selective pressure having resulted in a 'minimal' serpin that lacks the regions not needed for structural integrity or inhibitory activity. The docking model provides an explanation for the selectivity of CrmA. Our demonstration that engineering optimal substrate recognition sequences into the CrmA reactive-site loop fails to generate a good caspase 3 inhibitor is consistent with the docking model.

3-Amidinophenylalanine-based inhibitors of urokinase.

Synthesis and anti-uPA activity of a series of Nalpha-triisopropyl-phenylsulfonyl-protected 3-amidinophenylalanine amides are described. We have explored SAR around the C-terminal amide part for inhibition of uPA, plasmin and trypsin. Modification of the amide part has been found to affect potency but not selectivity. With a Ki of 0.41 microM 2r-L is one of the most potent uPA inhibitors described so far. The X-ray crystal structure of 2r-L was solved in complex with trypsin, superimposed with uPA and the results suggest an unique binding mode of this inhibitor type.

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines.

Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an Nalpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-is onipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [Nalpha-(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylal anyl-piperid ide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (Ki = 0. 50 +/- 0.14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 +/- 0.08 pM). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human alpha-thrombin was solved and confirmed the expected bivalent binding mode.

An active zymogen: unravelling the mystery of tissue-type plasminogen activator.

In contrast to almost all other proteinases, human tissue-type plasminogen activator (tPA) is also proteolytically active in its zymogen or single-chain form. The closely related plasminogen activator isolated from vampire bat saliva (vPA) acts exclusively in the single-chain form, lacking the requisite cleavage site for proteolytic activation. Recent structural studies on the proteolytic domains of vPA and human tPA in two- and single-chain forms reveal the mechanism of this anomalous activity. The PA-catalyzed proteolytic conversion of plasminogen to plasmin, responsible for the initiation of fibrinolysis, is fibrin-dependent; comparative structural analysis of the plasminogen activators provides clues as to the role of fibrin as cofactor.

Structural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase.

The trypsin-like serine proteinase superfamily contains a number of potential therapeutic targets, many of which are unsuitable for routine X-ray crystallographic studies. We have cocrystallized a selection of benzamidine-based inhibitors with bovine trypsin and solved their structures to a resolution of up to 1.7 A. Despite similar chemical formulas, the inhibitors exhibit a range of diverse binding modes that reflect their inhibitory spectra against the serine proteinases trypsin, thrombin, factor Xa, tissue-type plasminogen activator (tPA) and urokinase (uPA). In contrast to the compact folded conformations of thrombin inhibitors which allow optimal binding in the well-defined hydrophobic S2/S4 pocket of thrombin, those effective against factor Xa exhibit an extended conformation that allows occupation of the S3/S4 region, where hydrophobic and electrostatic interactions can stabilize the conformation. One group of inhibitors containing an N-terminal 2,4, 6-triisopropylphenylsulfonyl (TIPPS) moiety show little or no penetration into the S3/S4 subsites of trypsin. These latter sites are occluded in uPA, explaining why this class of compounds is effective against uPA. Despite presenting an extensive hydrophobic surface toward the solvent, the Ki values for TIPPS-containing compounds against trypsin is in the range 10(-7) to 10(-8) M. Comparison of the binding of a bis-benzamidine inhibitor in trypsin and tPA indicate that a shift in potency can be induced by relatively minor changes in binding mode. Implications for the inhibition of these proteinases are discussed.

Catalytic domain structure of vampire bat plasminogen activator: a molecular paradigm for proteolysis without activation cleavage.

The saliva of the blood-eating vampire bat Desmodus rotundus contains plasminogen activators (PAs) that maintain the fluidity of the prey's blood by activating plasminogen and dissolving developing fibrin clots. D. rotundus salivary PAs (DSPAs) are composed of evolutionarily conserved domains reminiscent of human tissue-type PA (tPA), but their catalytic domain lacks a plasmin-sensitive "activation cleavage site". Despite this, all DSPAs are intrinsically active and enormously stimulated in the presence of fibrin. The recombinant catalytic domain of DSPAalpha1 has been crystallized in a covalent complex with Glu-Gly-Arg-chloromethyl ketone and its structure solved at 2.9 A resolution. The structure is similar to that of activated two-chain human tPA. Despite its single-chain status, the activation domain is observed in an enzymatically active conformation, with a functional substrate binding site and active site accommodating the peptidylmethylene inhibitor. The activation pocket, which normally receives the N-terminal Ile16, is occupied by the side chain of Lys156, whose distal ammonium group makes an internal salt bridge with the carboxylate group of Asp194. Lys156 is in a groove shielded from the bulk solvent by the intact "activation loop" (Gln10-Phe21), favoring Lys156-Asp194 salt bridge formation and stabilization of a functional substrate binding site. Together with the characteristic 186 insertion loop, the activation loop could act as a switch, effecting full single-chain enzymatic activity upon binding to fibrin.

Structural mapping of the active site specificity determinants of human tissue-type plasminogen activator. Implications for the design of low molecular weight substrates and inhibitors.

The recent structure determination of the catalytic domain of tissue-type plasminogen activator (tPA) suggested residue Arg174 could play a role in P3/P4 substrate specificity. Six synthetic chromogenic tPA substrates of the type R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N-terminal protection group, were synthesized to test this property. Although changing the residue Xaa (in its L or D form) at position P3 from the hydrophobic Phe to an acidic residue, Asp or Glu, gave no improvement in catalytic efficiency, comparative analysis of the substrates indicated a preference for an acidic substituent occupying the S3 site when the S4 site contains a hydrophobic or basic moiety. The 2.9 A structure determination of the catalytic domain of human tPA in complex with the bis-benzamidine inhibitor 2, 7-bis-(4-amidinobenzylidene)-cycloheptan-1-one reveals a three-site interaction, salt bridge formation of the proximal amidino group of the inhibitor with Asp189 in the primary specificity pocket, extensive hydrophobic surface burial, and a weak electrostatic interaction between the distal amidino group of the inhibitor and two carbonyl oxygens of the protein. The latter position was previously occupied by the guanidino group of Arg174, which swings out to form the western edge of the S3 pocket. These data suggest that the side chain of Arg174 is flexible, and does not play a major role in the S4 specificity of tPA. On the other hand, this residue would modulate S3 specificity, and may be exploited to fine tune the specificity and selectivity of tPA substrates and inhibitors.

Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single-chain human tPA.

Tissue type plasminogen activator (tPA) is the physiological initiator of fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then degrades fibrin with relatively broad specificity. Unlike other chymotrypsin family serine proteinases, tPA is proteolytically active in a single-chain form. This form is also preferred for therapeutic administration of tPA in cases of acute myocardial infarction. The proteolytic cleavage which activates most other chymotrypsin family serine proteinases increases the catalytic efficiency of tPA only 5- to 10-fold. The X-ray crystal structure of the catalytic domain of recombinant human single-chain tPA shows that Lys156 forms a salt bridge with Asp194, promoting an active conformation in the single-chain form. Comparisons with the structures of other serine proteinases that also possess Lys156, such as trypsin, factor Xa and human urokinase plasminogen activator (uPA), identify a set of secondary interactions which are required for Lys156 to fulfil this activating role. These findings help explain the anomalous single-chain activity of tPA and may suggest strategies for design of new therapeutic plasminogen activators.

Tissue-type plasminogen activator: variants and crystal/solution structures demarcate structural determinants of function.

NMR and crystal structure of many components of tissue-type plasminogen activator (t-PA) are now available: the finger-EGF pair and the kringle-2 domain structures have been solved, as have the proteolytic domains of vampire bat PA and human t-PA in two- and single-chain forms. These structures confirm the trypsin-like arrangement of the proteolytic domain of t-PA and show how surface loops near the catalytic centre contribute to the narrow specificity of t-PA. Together with mutational experiments, they identify the Lys156 sidechain as a cause of the amidolytic activity of single-chain t-PA, as it can provide a substitute salt bridge partner for Asp194 in the absence of the Ile16 N terminus of the two-chain form. These new findings provide new ideas for the design of PA variants with improved therapeutic properties.

Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors.

The solution structure and the disulfide pairings of a 36-residue proteinase inhibitor isolated from the insect Locusta migratoria have been determined using NMR spectroscopy and simulated annealing calculations. The peptide, termed PMP-C, was previously shown to inhibit bovine alpha-chymotrypsin as well as human leukocyte elastase, and was also found to block high-voltage-activated Ca2+ currents in rat sensory neurones. PMP-C has a prolate ellipsoid shape and adopts a tertiary fold hitherto unobserved in the large group of small "canonical" proteinase inhibitors. The over-all fold consists mainly of three strands arranged in a right-handed twisted, antiparallel, beta-sheet that demarcates a cavity, together with a linear amino-terminal segment oriented almost perpendicular to the three strands of the beta-sheet. Inside the cavity a phenyl ring constitutes the centre of a hydrophobic core. The proteinase binding loop is located in the carboxy-terminal part of the molecule, between two cysteine residues involved in disulfide bridges. Its conformation resembles that found in other small canonical proteinase inhibitors. A comparison of PMP-C structure with the recently published solution structure of the related peptide PMP-D2 shows that the most significant differences are complementary changes involved in the stabilization of similar folds. This comparison led us to review the structure of PMP-D2 and to identify two salt bridges in PMP-D2.