Human indoleamine 2,3-dioxygenase (IDO) catalyzes the cleavage of the pyrrol ring of L-Trp and incorporates both atoms of a molecule of oxygen (O 2). Here we report on the x-ray crystal structure of human IDO, complexed with the ligand inhibitor 4-phenylimidazole and cyanide. The overall structure of IDO shows two ␣-helical domains with the heme between them. A264 of the flexible loop in the heme distal side is in close proximity to the iron. A mutant analysis shows that none of the polar amino acid residues in the distal heme pocket are essential for activity, suggesting that, unlike the heme-containing monooxygenases (i.e., peroxidase and cytochrome P450), no protein group of IDO is essential in dioxygen activation or proton abstraction. These characteristics of the IDO structure provide support for a reaction mechanism involving the abstraction of a proton from the substrate by iron-bound dioxygen. Inactive mutants (F226A, F227A, and R231A) retain substratebinding affinity, and an electron density map reveals that 2-(Ncyclohexylamino)ethane sulfonic acid is bound to these residues, mimicking the substrate. These findings suggest that strict shape complementarities between the indole ring of the substrate and the protein side chains are required, not for binding, but, rather, to permit the interaction between the substrate and iron-bound dioxygen in the first step of the reaction. This study provides the structural basis for a heme-containing dioxygenase mechanism, a missing piece in our understanding of heme chemistry.iron ͉ kynurenine ͉ tryptophan ͉ x-ray crystallography O xygenases (1) are metal-containing enzymes that catalyze the incorporation of a molecule of oxygen (O 2 ) into the substrate, and thus play a crucial role in the metabolism and synthesis of a variety of biological substances. Two types of oxygenase are currently known: monooxygenases (scheme I) and dioxygenases (scheme II):In the 1950s and 1960s, Hayaishi and coworkers reported that two heme-containing dioxygenases, indoleamine 2,3-dioxygenase (IDO) (2) and tryptophan 2,3-dioxygenase (TDO) (3), catalyze the initial and rate-limiting step of L-Trp catabolism in the kynurenine (Kyn) pathway (4). This step involves the oxidative cleavage of the 2,3 double bond in the indole moiety of L-Trp, resulting in the production of N-formyl Kyn. Increased levels of the Kyn pathway metabolites quinolinic acid and 3-hydroxykynurenine (3OHKyn) have been observed in a number of neurological or psychiatric disorders. L-Trp-derived UV filters (Kyn and 3OHKyn glucoside) can bind to the lens protein and appear to be mainly responsible for the nuclear cataract (5). L-Trp also serves as a precursor for the synthesis of the neurotransmitter serotonin and the hormone melatonin. IDO exhibits a broader substrate specificity than TDO, because the former can degrade indoleamines, including L-Trp, D-Trp, serotonin, melatonin, and tryptamine (6). In addition to its role as a L-Trp-catabolizing enzyme, IDO is involved in the immunoregulating system [review by Mellor and M...

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causeshemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or a RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures (i.e. monomer, dimer, and octamer) can interact with PScontaining membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys-224, 225 and Lys-274, 275). Substitutions of these residues with alanine reduced PSvesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest http://www.jbc.org/cgi

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.