The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 Å crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated ␤-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed ␤-propeller with open Velcro topology and the C-terminal ␣͞␤-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the ␤-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu-Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P 2-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.serine protease ͉ oxyanion hole ͉ substrate channeling ͉ drug design ͉ diabetes mellitus

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor Vllla. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-21-Arg-31 chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 1100, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fiX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-fVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVlIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.Human factor IX (fIX) is secreted as a 415-residue single-chain molecule into the blood, where it is activated to fIXa by proteolytic cleavage (1-3). A single cleavage at Arg-180-Val-181 [Arg-181-Ile-182 in porcine flX (refs. 4-6; P.L., unpublished data)], corresponding to residues 15 and 16 in chymotrypsinogen numbering (hereafter denoted with the prefix c) generates active form fIXaa, while a second cleavage removes segment Ala-146-Arg-180 to generate the physiological active form fIXaf3 (7,8). The N-terminal light chain (145 residues) and the C-terminal heavy chain (235 residues) are disulfide linked via Cys-132-Cys-289(c122). The light chain consists of several modules, which also reflect the exon structure (9): the N-terminal Gla module (residues 1-38; Gla refers to Cy carboxylated glutamic acid residues) followed by its hydrophobic helix (39-46), two epidermal growth facto...

The cysteine protease legumain plays important functions in immunity and cancer at different cellular locations, some of which appeared conflicting with its proteolytic activity and stability. Here, we report crystal structures of legumain in the zymogenic and fully activated form in complex with different substrate analogs. We show that the eponymous asparagine-specific endopeptidase activity is electrostatically generated by pH shift. Completely unexpectedly, the structure points toward a hidden carboxypeptidase activity that develops upon proteolytic activation with the release of an activation peptide. These activation routes reconcile the enigmatic pH stability of legumain, e.g., lysosomal, nuclear, and extracellular activities with relevance in immunology and cancer. Substrate access and turnover is controlled by selective protonation of the S1 pocket (K M ) and the catalytic nucleophile (k cat ), respectively. The multibranched and context-dependent activation process of legumain illustrates how proteases can act not only as signal transducers but as decision makers.allostery | context-dependent activities | death domain | k cat -substrate specificity | electrostatic stability switch

The last years have seen a steady increase in our understanding of legumain biology that is driven from two largely uncoupled research arenas, the mammalian and the plant legumain field. Research on legumain, which is also referred to as asparaginyl endopeptidase (AEP) or vacuolar processing enzyme (VPE), is slivered, however. Here we summarise recent important findings and put them into a common perspective. Legumain is usually associated with its cysteine endopeptidase activity in lysosomes where it contributes to antigen processing for class II MHC presentation. However, newly recognized functions disperse previously assumed boundaries with respect to their cellular compartmentalisation and enzymatic activities. Legumain is also found extracellularly and even translocates to the cytosol and the nucleus, with seemingly incompatible pH and redox potential. These different milieus translate into changes of legumain's molecular properties, including its (auto-)activation, conformational stability and enzymatic functions. Contrasting its endopeptidase activity, legumain can develop a carboxypeptidase activity which remains stable at neutral pH. Moreover, legumain features a peptide ligase activity, with intriguing mechanistic peculiarities in plant and human isoforms. In pathological settings, such as cancer or Alzheimer's disease, the proper association of legumain activities with the corresponding cellular compartments is breached. Legumain's increasingly recognized physiological and pathological roles also indicate future research opportunities in this vibrant field.

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The 3.0-Å resolution x-ray structure of human des-Glacoagulation factor Xa (fXa) has been determined in complex with the synthetic inhibitor DX-9065a. The binding geometry is characterized primarily by two interaction sites: the naphthamidine group is fixed in the S1 pocket by a typical salt bridge to Asp-189, while the pyrrolidine ring binds in the unique aryl-binding site (S4) of fXa. Unlike the large majority of inhibitor complexes with serine proteinases, Gly-216 (S3) does not contribute to hydrogen bond formation. In contrast to typical thrombin binding modes, the S2 site of fXa cannot be used by DX-9065a since it is blocked by Tyr-99, and the arylbinding site (S4) of fXa is lined by carbonyl oxygen atoms that can accommodate positive charges. This has implications for natural substrate recognition as well as for drug design.Hemostasis is the blood clotting process that, when functioning properly, occurs when an injury to the vasculature leads to a series of vasculomotor and cellular reactions and the activation of the blood coagulation cascade. The latter process is initiated via the extrinsic pathway, leading first to thrombin activation and then massively amplified thrombin activation due to the positive feedback of the intrinsic pathway (1). Both extrinsic and intrinsic pathways merge at the factor X activation step. An imbalance between these clotting processes, clotting inactivation processes (protein C inactivation of hemostasis cofactors), and thrombolytic processes (tissue plasminogen activator, plasminogen) can lead to thrombotic or bleeding disorders. Antithrombotics include inhibitors of thrombin, factor Xa and factor IXa, factors involved in both the extrinsic and intrinsic pathways (2). As evidence accumulates that thrombin has other important functions in cellular (3, 4) and neurological (5-10) processes, new synthetic anticoagulants increasingly target factor Xa. Daiichi published the first tight binding (K i ϭ 41 nM), specific inhibitor of fXa, 1 DX-9065a (11-13). We present here the crystal structure of the factor Xa⅐DX-9065a complex. The inhibitor binds in the active site in an extended conformation, which was expected from earlier studies (14, 15). Both hydrophobic and electrostatic interactions characterize the complex formation, which is also accompanied by local rearrangements in the active site of fXa. Considering these subtle interactions and the unpredictable ligand-induced motions involved in binding, there is clearly a need for a series of fXa-ligand complex structures to provide adequate information for structure-based drug design and an understanding of how fXa recognizes physiological substrates.EXPERIMENTAL PROCEDURES fX was isolated from human plasma; des-Gla-fX was produced via chymotryptic cleavage (removing amino acids L1-L44; chymotrypsinogen numbering is used for the catalytic domain; the sequential fX numbering, which is used for the light chain, will be indicated by the prefix "L"). Subsequently, fX was activated with the purified factor X activator from Russell's vip...

One of the most precisely regulated processes in living cells is intracellular protein degradation. The main component of the degradation machinery is the 20S proteasome present in both eukaryotes and prokaryotes. In addition, there exist other proteasome-related protein-degradation machineries, like HslVU in eubacteria. Peptides generated by proteasomes and related systems can be used by the cell, for example, for antigen presentation. However, most of the peptides must be degraded to single amino acids, which are further used in cell metabolism and for the synthesis of new proteins. Tricorn protease and its interacting factors are working downstream of the proteasome and process the peptides into amino acids. Here, we summarise the current state of knowledge about protein-degradation systems, focusing in particular on the proteasome, HslVU, Tricorn protease and its interacting factors and DegP. The structural information about these protein complexes opens new possibilities for identifying, characterising and elucidating the mode of action of natural and synthetic inhibitors, which affects their function. Some of these compounds may find therapeutic applications in contemporary medicine.

The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.