Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) play a central role in tryptophan metabolism and are involved in many cellular and disease processes. Here we report the crystal structure of human TDO (hTDO) in a ternary complex with the substrates L-Trp and O2 and in a binary complex with the product N-formylkynurenine (NFK), defining for the first time the binding modes of both substrates and the product of this enzyme. The structure indicates that the dioxygenation reaction is initiated by a direct attack of O2 on the C2 atom of the L-Trp indole ring. The structure also reveals an exo binding site for L-Trp, located ~42 Å from the active site and formed by residues conserved among tryptophan-auxotrophic TDOs. Biochemical and cellular studies indicate that Trp binding at this exo site does not affect enzyme catalysis but instead it retards the degradation of hTDO through the ubiquitin-dependent proteasomal pathway. This exo site may therefore provide a novel L-Trp-mediated regulation mechanism for cellular degradation of hTDO, which may have important implications in human diseases.

tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3'-end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the beta-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for the coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein, we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops, and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V, and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reduces efficiency by approximately 1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO. In addition, in hIDO1, the indoleamine group of the substrate Trp is H-bonded to S167 through a bridging water, while that in hTDO is directly H-bonded to H76. Here we show that Trp binding to the Si site or the mutation of S167 to histidine in hIDO1 retards its turnover activity and that the inhibited activity can be rescued by an effector, 3-indole ethanol (IDE). Kinetic studies reveal that the inhibited activity introduced by Trp binding to the Si site is a result of retarded recombination of the ferryl moiety with Trp epoxide to form NFK and that IDE reverses the effect by preventing Trp from binding to the Si site. In contrast, the abolished activity induced by the S167H mutation is primarily a result of ∼5000-fold reduction in the O binding rate constant, possibly due to the blockage of a ligand delivery tunnel, and that IDE binding to the Si site reverses the effect by reopening the tunnel. The data offer new insights into structure-based design of hIDO1-selective inhibitors.

IntroductionThe human ␤-and ␣-globin gene loci are developmentally regulated and are arrayed spatially in the order in which they are expressed developmentally, respectively, 5Ј-⑀-G ␥-A ␥-␤-3Ј and 5Ј--␣-␣-3Ј. Fetal hemoglobin (HbF), an ␣ 2 ␥ 2 -tetramer, predominates from week 8 of gestation through birth, after which it is gradually superseded by adult hemoglobin (HbA), an ␣ 2 ␤ 2 -tetramer. HbF is detectable, at approximately 1%, during adult life. The molecular mechanisms underlying the ␥-to ␤-globin switch during development are not fully understood, but are of compelling interest because a persistence of HbF in adults, whether genetic or pharmacologic in origin, is ameliorative in adult ␤-globin gene disorders such as sickle cell anemia or ␤-thalassemia. 1,2 Intermediaries of mammalian metabolism, such as the shortchain fatty acids (SCFAs) butyrate and propionate, are important fuels during fetal life and, when elevated, are implicated in the delayed fetal-to-adult hemoglobin switch in infants of diabetic mothers (butyrate 3 ) and in the elevated HbF levels seen in children with inherited disorders of branched-chain amino acid metabolism (eg, propionic acidemia 4 or ␤-ketothiolase deficiency 5 ). Importantly, therapeutic trials of butyrate in patients with ␤-globin gene disorders have increased ␥-globin gene expression and HbF levels. [6][7][8] Studies of the molecular and cellular effects of SCFAs in erythroid cells have been constrained by the limited number of experimental models currently available. We were interested in finding additional models in which to study the effects of SCFAs on erythropoiesis and on erythroid gene expression. We evaluated the effects of SCFAs on murine erythropoeisis in primitive and definitive erythroid precursor cells from transgenic mice that had endogenous elevations of SCFAs, and in definitive erythroid precursor cells from wt mice and human ␤-globin gene locuscontaining transgenic mice. These studies were undertaken with the expectation that a biologically relevant, primary, definitive erythroid precursor cell model would be an excellent place in which to study the pleiotropic effects of SCFAs on erythropoiesis.The murine ␤-globin gene locus, like the human, is developmentally regulated. ␤ H1 expression is detectable early and persists through embryonic day (E) 12 to 13, while ⑀ y expression is detected later and is present through E13 through 16. 9,10 These embryonic ␤-type globin genes, and the embryonic ␣-like -globin gene, are expressed in large, slowly enucleating, primitive erythroid (EryP) cells from the murine yolk sac. Adult ␤-type globin genes, ␤ Adult , comprise the ␤ maj and ␤ min genes (from the ␤-diffuse haplotype found in most strains) or the ␤ S and ␤ T genes (from the ␤-single haplotype found in the C57/black 6 strain). ␤ Adult and ␣ are expressed primarily in small, rapidly enucleated, definitive erythroid (EryD) cells that arise from the erythroid fetal liver through late gestation and from the adult bone marrow and, in anemic animals, from the adult e...