Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygenbinding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.cancer ͉ heme enzymes ͉ immunomodulation ͉ indoleamine 2,3-dioxygenase T ryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze the oxidative cleavage of the L-tryptophan (L-Trp) pyrrole ring, the first and rate-limiting step in L-Trp catabolism through the kynurenine pathway (1-3). In addition, IDO has been implicated in a diverse range of physiological and pathological conditions, including suppression of T cell proliferation, maternal tolerance to allogenic fetus, and immune escape of cancers (4-8), and is an attractive target for drug discovery against cancer and autoimmune and other diseases (2, 9-12).Despite catalyzing identical biochemical reactions (Fig. 1a), the sequence similarity between TDO and IDO is extremely low. An alignment of their sequences is only possible based on their structures, which suggests a sequence identity of 10% between them (Fig. 1b). In comparison, Xanthomonas campestris TDO shares 34% sequence identity with human TDO (Fig. 1b), demonstrating the remarkable evolutionary conservation of this enzyme. TDO is a homotetrameric enzyme and is highly specific for L-Trp and related derivatives such as 6-fluoro-Trp as the substrate. In comparison, IDO is monomeric, and shows activity toward a larger collection of substrates, including L-Trp, Dtryptophan (D-Trp), serotonin, and tryptamine (3), although the K m for D-Trp is Ϸ100-fold higher than that for L-Trp (13). The structure of human IDO in the catalytically inactive, ferric [Fe(III)]-heme state in complex with the 4-phenylimidazole inhibitor has recently been reported (14). Although this structure gave information about important active site residues, the inhibitor is coordinat...

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-␤ mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3 end processing of all cellular pre-mRNAs. Here we report the 1.95-Å resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-␤ pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.antiviral drug discovery ͉ bird flu ͉ vaccine engineering ͉ virology ͉ X-ray crystallography

The world is currently undergoing a pandemic caused by an H1N1 influenza A virus, the so-called 'swine flu'. The H5N1 ('bird flu') influenza A viruses, now circulating in Asia, Africa and Europe, are extremely virulent in humans, although they have not so far acquired the ability to transfer efficiently from human to human. These health concerns have spurred considerable interest in understanding the molecular biology of influenza A viruses. Recent structural studies of influenza A virus proteins (or fragments) help enhance our understanding of the molecular mechanisms of the viral proteins and the effects of drug resistance to improve drug design. The structures of domains of the influenza RNA-dependent RNA polymerase and the nonstructural NS1A protein provide opportunities for targeting these proteins to inhibit viral replication.Influenza A viruses are responsible for sporadic pandemics that usually cause higher mortality rates than seasonal influenza epidemics. The most severe pandemic occurred in 1918, resulting in approximately 40 million deaths worldwide 1 . There were also pandemics in 1957 and 1968. In fact, we are currently in the midst of a pandemic caused by a virus originating in swine, the 2009 H1N1 virus or 'swine flu' 2,3 . In addition, H5N1 viruses ('bird flu'), which are also currently circulating, are extremely virulent in humans but have not yet acquired the ability for efficient human-to-human transmission (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_07_01/en/ index.html).Influenza A viruses infect a wide range of avian and mammalian hosts, unlike influenza B viruses, which infect only humans. The envelope of influenza A viruses contains two different surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) 4,5 . Influenza A viruses are categorized into antigenic HA and NA subtypes: 16 HA (H1-H16) and 9 NA (N1-N9) antigenic subtypes have been identified so far. Swine flu is an H1N1 virus because it contains a H1 subtype HA and a N1 subtype NA. The major influenza A subtypes that have infected © 2010 Nature America, Inc. All rights reserved.Correspondence should be addressed to K.D. (kalyan@cabm.rutgers.edu).. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Here we present a structural-biology perspective on the existing and emerging molecular targets for anti-influenza drugs. The viral proteins are discussed in the order of their primary functions in the influenza A life cycle as outlined in the schematic representation in Figure 1. NIH Public Access HemagglutininHA molecules, which form trimers, attach the virus to sialic acid receptors on the cell surface and mediate the release of viral ribonucleoprotein particles (vRNPs) into the cytoplasm. A newly synthesized ~70-kDa HA is cleaved into HA1 and HA2, which are disulfide linked ( NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript co...