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...

SUMMARY UNC119 is widely expressed among vertebrates and invertebrates. Here we report that UNC119 recognized the acylated N-terminus of the rod photoreceptor transducin α-subunit (Tα) as well as C. elegans G proteins Odr-3 and Gpa-13. The crystal structure of human UNC119 at 1.95 Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. Co-crystallization of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound TαGTP inhibiting its GTPase activity, thereby providing a stable UNC119-TαGTP complex that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation. UNC119 deletion in both mouse and C. elegans lead to G protein mislocalization. These results establish UNC119 as a novel Gα-subunit cofactor that is essential for G-protein trafficking in sensory cilia.

With over 60,000 protein structures available in the Protein Data Bank, it is frequently possible use one of them to obtain starting phase information and to solve new crystal structures. Molecular replacement1–4 procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods5–8, have allowed the rapid solution of large numbers of protein structures. Despite extensive work9–14, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modeling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modeling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction datasets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction datasets of better than 3.2Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 Å. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.A ctin is a ubiquitous 43-kDa adenine nucleotide-binding protein involved in a wide range of fundamental processes in eukaryotic cells, including cell motility (1), cytokinesis (2), vesicle transport (3, 4), and the establishment and maintenance of cell morphology (5, 6). Actin is also an essential component of numerous specialized structures in higher eukaryotes, such as the intestinal brush border (7,8) and stereocilia (9, 10). The ability of actin to participate in these diverse processes is a consequence of the dynamic and polymorphic nature of the filamentous actin (F-actin) assemblies that are formed in vivo.Central to understanding cytoskeletal regulation is the contribution of the adenine nucleotide to actin structure, function, and dynamics. Bound nucleotide is required to stabilize the actin monomer under physiological conditions, because nucleotidefree actin denatures at a rate of 0.2 s Ϫ1 (11). ATP hydrolysis occurs subsequent to monomer addition to the growing filament, thus ''marking'' the oldest part of the filament (i.e., ADP-actin), and may serve as a timing mechanism for filament disassembly, analogous to the role of GTP hydrolysis in G proteins (12, 13). Furthermore, several regulatory proteins (e.g., profilin, cofilin, and thymosin ␤-4) show specificity for either ATP-or ADPactin, which is thought to be an important element of cytoskeletal regulation (12). Actin dynamics also depends on the identity of the bound metal-nucleotide complex (14-21).The structure of the actin monomer has been known for over a decade, and the structures of mammalian Ca 2ϩ -ATP actins in complex with DNase I (22), profilin (23), gelsolin segment-1 (24), and gelsolin segments-4-6 (25) are available. An orthorhombic crystal form of the Ca 2ϩ -ATP Dictyostelium actin͞ gelsolin segment-1 structure has also been reported (26). These structures highlighted specific regulatory interactions and revealed the remarkable architectural similarity between actin and a wide range of proteins (27), including the heat shock cognate ATPases (e.g., HSC70) and sugar kinases (e.g., hexokinase) (28-3...

Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion-holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by sidechains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (kcat/KM) of 400 M−1s−1 for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

An antiparallel actin dimer has been proposed to be an intermediate species during actin filament nucleation. We now show that latrunculin A, a marine natural product that inhibits actin polymerization, arrests polylysine-induced nucleation at the level of an antiparallel dimer, resulting in its accumulation. These dimers, when composed of pyrene-labeled actin subunits, give rise to a fluorescent excimer, permitting detection during polymerization in vitro. We report the crystallographic structure of the polylysine-actin-latrunculin A complex at 3.5-Å resolution. The non-crystallographic contact is consistent with a dimeric structure and confirms the antiparallel orientation of its subunits. The crystallographic contacts reveal that the mobile DNase I binding loop of one subunit of a symmetry-related antiparallel actin dimer is partially stabilized in the interface between the two subunits of a second antiparallel dimer. These results provide a potential explanation for the paradoxical nucleation of actin filaments that have exclusively parallel subunits by a dimer containing antiparallel subunits.Actin filament nucleation occurs very slowly de novo, but it occurs rapidly as a necessary step in actin-based motility (1). The formation of a dimer from monomeric subunits is the most thermodynamically unfavorable nucleation step with an estimated equilibrium dissociation constant of 4.6 M (in contrast to 0.6 mM for conversion of dimer to trimer) in a recent molecular dynamic simulation of nucleation (2). The formation of an effective nucleus may be accelerated in vivo by an actin-binding protein such as gelsolin, which can stabilize dimeric actin, or by a protein complex such as Arp2/3 that is thought to contain two actin-like molecules constrained in an orientation that promotes nucleation (3, 4). Antiparallel actin dimers have been identified as a precursor to actin filament polymerization by covalent cross-linking during polymerization induced with divalent cations (5). A gelsolin-actin complex capable of nucleating filament growth at the slow growing, pointed end of filaments has also been shown by covalent cross-linking to contain two actin subunits in the antiparallel configuration (6). The assumption of an antiparallel configuration of subunits is based on evidence that Cys-374 in the C terminus of actin is the only residue involved in the cross-linking reaction. In contrast, when polymerization is complete, intrafilament cross-linking yields a parallel dimer. More recently, electron microscopy has revealed that newly formed actin filaments show evidence of incorporation of antiparallel dimers. This incorporation results in a branched filament network, implying that the dimers have nucleating activity (7). Interestingly, analysis of a Listeria model of cell motility using high-resolution laser tracking provides evidence that filaments elongate in 5.4 nm steps, consistent with in vivo incorporation of dimeric actin (8).In the current work, we provide evidence that polylysine nucleates actin polymerization by e...

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