The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 Å), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYYbound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 Å apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.C-C coupling ͉ cyclopeptide

We report the crystal structure of a soluble form of human urokinase-type plasminogen activator receptor (uPAR/ CD87), which is expressed at the invasive areas of the tumor-stromal microenvironment in many human cancers. The structure was solved at 2.7 Å in association with a competitive peptide inhibitor of the urokinasetype plasminogen activator (uPA)-uPAR interaction. uPAR is composed of three consecutive three-finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA-uPAR complex. This model reveals that the receptorbinding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA-focalized proteolysis on the cell surface and control cell adhesion and migration.

A novel heterodimeric three-finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three-finger protein fold, typical for "nonconventional" toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three-finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon-specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC(50)=10 nM), comparable to alpha-bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three-finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.

Human placental alkaline phosphatase (PLAP) is one of three tissue-specific human APs extensively studied because of its ectopic expression in tumors. The crystal structure, determined at 1.8-Å resolution, reveals that during evolution, only the overall features of the enzyme have been conserved with respect to Escherichia coli. The surface is deeply mutated with 8% residues in common, and in the active site, only residues strictly necessary to perform the catalysis have been preserved. Additional structural elements aid an understanding of the allosteric property that is specific for the mammalian enzyme (Hoylaerts, M. F., Manes, T., and Millá n, J. L. (1997) J. Biol. Chem. 272, 22781-22787). Allostery is probably favored by the quality of the dimer interface, by a long N-terminal ␣-helix from one monomer that embraces the other one, and similarly by the exchange of a residue from one monomer in the active site of the other. In the neighborhood of the catalytic serine, the orientation of Glu-429, a residue unique to PLAP, and the presence of a hydrophobic pocket close to the phosphate product, account for the specific uncompetitive inhibition of PLAP by L-amino acids, consistent with the acquisition of substrate specificity. The location of the active site at the bottom of a large valley flanked by an interfacial crown-shaped domain and a domain containing an extra metal ion on the other side suggest that the substrate of PLAP could be a specific phosphorylated protein.

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Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use. Introduction:We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1 Ϫ/Ϫ and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site. Materials and Methods:We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 Å radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors L-homoarginine (L-hArg), levamisole, theophylline, and L-phenylalanine. Results and Conclusions:We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.

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The high affinity interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) represents one of the key regulatory steps in cell surface-associated plasminogen activation. On the basis on our crystal structure solved for uPAR in complex with a peptide antagonist, we recently proposed a model for the corresponding complex with the growth factor-like domain of uPA (Llinas et al. (2005) EMBO J. 24, 1655-1663). In the present study, we provide experimental evidence that consolidates and further develops this model using data from a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were determined by surface plasmon resonance. This complete alanine scanning of uPAR highlighted the involvement of 20 surface-exposed side chains in this interaction. Mutations causing ⌬⌬G > 1 kcal/mol for the uPA interaction are all located within or at the rim of the central cavity uniquely formed by the assembly of all three domains in uPAR, whereas none are found outside this crevice. Identification of specific cross-linking sites in uPAR and pro-uPA enabled us to build a model of the uPAR⅐uPA complex in which the kringle domain of uPA was positioned by the constraints established by the range of these cross-linkers. The nature of this interaction is predominantly hydrophobic and highly asymmetric, thus emphasizing the importance of the shape and size of the central cavity when designing low molecular mass antagonists of the uPAR/uPA interaction.The urokinase-type plasminogen activator receptor (uPAR) 2 is a glycosylphosphatidylinositol-anchored membrane glycoprotein (1) that has a primary role in focalizing plasminogen activation at the cell surface through its specific high affinity interaction with the urokinase-type plasminogen activator (uPA). Besides facilitating the generation of plasmin activity in the vicinity of uPAR-expressing cells, which is directly or indirectly involved in remodeling of the extracellular matrix (2, 3), the uPAR/pro-uPA interaction also assists in regulating other aspects of cell adhesion and migration. Among these molecular processes is the direct interaction with matrix-deposited vitronectin (4); the modulation of integrin function, in particular ␣ M ␤ 2 , ␣ 5 ␤ 1 , and ␣ 3 ␤ 1 (5-8); and the activation of the chemotactic FPRL1 receptor (9). As an increased expression level of uPAR is often found in the invasive areas of various human cancers and correlates with poor prognosis (10), the uPAR/uPA interaction and uPA catalytic activity are considered relevant molecular targets for drug development (11-13). Intervention strategies developed for targeting the uPAR/uPA interaction with a view to cancer therapy include recombinant fusion proteins containing the receptor-binding module of uPA (14,...