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Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 Å. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.

Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineered artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, to examine the activity of effector domains from natural splicing factors and to modulate splicing of an endogenous human gene, Bcl-X, an anticancer target. The designer factor targeted to Bcl-X increased the amount of pro-apoptotic Bcl-xS splice isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common antitumor drugs. Our approach permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events.

Puf proteins bind RNA sequence specifically and regulate translation and stability of target mRNAs. A "code" for RNA recognition has been deduced from crystal structures of the Puf protein, human Pumilio1, where each of eight repeats binds an RNA base via a combination of three side chains at conserved positions. Here, we report the creation of seven soluble mutant proteins with predictably altered sequence specificity, including one that binds tightly to adenosine-uracil-rich element RNA. These data show that Pumilio1 can be used as a scaffold to engineer RNA-binding proteins with designed sequence specificity.

Aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (ATIC) is a bifunctional enzyme with folate-dependent AICAR transformylase and IMP cyclohydrolase activities that catalyzes the last two steps of purine biosynthesis. The AICAR transformylase inhibitors BW1540 and BW2315 are sulfamido-bridged 5,8-dideazafolate analogs with remarkably potent K(i) values of 8 and 6 nm, respectively, compared with most other antifolates. Crystal structures of ATIC at 2.55 and 2.60 A with each inhibitor, in the presence of substrate AICAR, revealed that the sulfonyl groups dominate inhibitor binding and orientation through interaction with the proposed oxyanion hole. These agents then appear to mimic the anionic transition state and now implicate Asn(431') in the reaction mechanism along with previously identified key catalytic residues Lys(266) and His(267). Potent and selective inhibition of the AICAR transformylase active site, compared with other folate-dependent enzymes, should therefore be pursued by further design of sulfonyl-containing antifolates.

Within de novo purine biosynthesis, the AICAR transformylase and IMP cyclohydrolase activities of the bifunctional enzyme ATIC convert the intermediate AICAR to the final product of the pathway, IMP. Identification of the AICAR transformylase active site and a proposed formyl transfer mechanism have already resulted from analysis of crystal structures of avian ATIC in complex with substrate and/or inhibitors. Herein, we focus on the IMPCH active site and the cyclohydrolase mechanism through comparison of crystal structures of XMP inhibitor complexes of human ATIC at 1.9 A resolution with the previously determined avian enzyme. This first human ATIC structure was also determined to ascertain whether any subtle structural differences, compared to the homologous avian enzyme, should be taken into account for structure-based inhibitor design. These structural comparisons, as well as comparative analyses with other IMP and XMP binding proteins, have enabled a catalytic mechanism to be formulated. The primary role of the IMPCH active site appears to be to induce a reconfiguration of the substrate FAICAR to a less energetically favorable, but more reactive, conformer. Backbone (Arg64 and Lys66) and side chain interactions (Thr67) in the IMPCH active site reorient the 4-carboxamide from the preferred conformer that binds to the AICAR Tfase active site to one that promotes intramolecular cyclization. Other backbone amides (Ile126 and Gly127) create an oxyanion hole that helps orient the formyl group for nucleophilic attack by the 4-carboxamide amine and then stabilize the anionic intermediate. Several other residues, including Lys66, Tyr104, Asp125, and Lys137', provide substrate specificity and likely enhance the catalytic rate through contributions to acid-base catalysis.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole-5'-phosphate, an intermediate for the lower ligand of cobalamin. In earlier studies it proved difficult to obtain the structure of CobT bound to NaMN because it is hydrolyzed in the crystal lattice in the absence of the second substrate DMB. In an effort to map the reaction pathway of this enzyme, NaMN was captured in the active site with the substrate analogs 4,5-dimethyl-1,2-phenylenediamine, 4-methylcatechol, indole, 3,4-dimethylaniline, 2,5-dimethylaniline, 3,4-dimethylphenol, and 2-amino-p-cresol. Structures of these complexes reveal that they exclude water molecules responsible for the hydrolysis from the active site. These structures, together with the early complexes with alpha-ribazole-5'-phosphate and DMB, provide a complete description of the reaction pathway. They demonstrate that the nicotinate moiety and phosphate do not appear to move significantly between reactants and products but that the aromatic base and ribose moiety each move approximately 1.2 A toward each other in the transformation. This study also reveals that, like many other nucleotide binding proteins, coordination of DMB is accompanied by a disorder-order transition in a surface loop. The structure of apo-CobT is also reported.

The evolution of biosynthetic pathways is difficult to reconstruct in hindsight; however, the structures of the enzymes that are involved may provide insight into their development. One enzyme in the cobalamin biosynthetic pathway that appears to have evolved from a protein with different function is L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica, which is structurally similar to histidinol phosphate aminotransferase [Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C., and Rayment, I. (2002) Biochemistry 41, 4798-4808]. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. To understand the relationship between this decarboxylase and the aspartate aminotransferase family to which it belongs, the structures of CobD in its apo state, the apo state complexed with the substrate, and its product external aldimine complex have been determined at 1.46, 1.8, and 1.8 A resolution, respectively. These structures show that the enzyme steers the breakdown of the external aldimine toward decarboxylation instead of amino transfer by positioning the carboxylate moiety of the substrate out of the plane of the pyridoxal ring and by placing the alpha-hydrogen out of reach of the catalytic base provided by the lysine that forms the internal aldimine. It would appear that CobD evolved from a primordial PLP-dependent aminotransferase, where the selection was based on similarities between the stereochemical properties of the substrates rather than preservation of the fate of the external aldimine. These structures provide a sequence signature for distinguishing between L-threonine-O-3-phosphate decarboxylase and histidinol phosphate aminotransferases, many of which appear to have been misannotated.

The three-dimensional structure of the pyridoxal 5'-phosphate (PLP)-dependent L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica is described here. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. The molecule is a molecular dimer where each subunit consists of a large and small domain. Overall the protein is very similar to the members of the family of aspartate aminotransferases. Indeed, the arrangement of the ligands surrounding the cofactor and putative substrate binding site are remarkably close to that observed in histidinol phosphate aminotransferase, which suggests that this latter enzyme might have been its progenitor. The only significant differences in structure occur at the N-terminus, which is approximately 12 residues shorter in CobD and does not form the same type of interdomain interaction common to other aminotransferases. CobD is unusual since within the aspartate aminotransferase subfamily of PLP-dependent enzymes the chemical transformations are substantially conserved, where the only exceptions are 1-aminocyclopropane-1-carboxylate synthase and CobD. Although there are a large number of PLP-dependent amino acid decarboxylases, these are generally larger and structurally distinct from the members of the aspartate aminotransferase subfamily of enzymes. The structure of CobD suggests that the chemical fate of the external aldimine can be redirected by modifications at the N-terminus of the protein. This study provides insight into the evolutionary history of the cobalamin biosynthetic pathway and raises the question of why most PLP-dependent decarboxylases are considerably larger enzymes.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrates, giving rise to a wide variety of lower ligands in cobamides. To understand the molecular basis for this lack of substrate specificity, the x-ray structures of CobT complexed with adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, p-cresol, and phenol were determined. Furthermore, adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, and 2-hydroxypurine were observed to react with NaMN within the crystal lattice and undergo the phosphoribosyl transfer reaction to form product. Significantly, the stereochemistries of all products are identical to those found in vivo. Interestingly, p-cresol and phenol, which are the lower ligand in Sporomusa ovata, bound to CobT but did not react with NaMN. This study provides a structural explanation for how CobT can phosphoribosylate most of the commonly observed lower ligands found in cobamides with the exception of the phenolic lower ligands observed in S. ovata. This is accomplished with minor conformational changes in the side chains that constitute the 5,6-dimethylbenzimidazole binding site. These investigations are consistent with the implication that the nature of the lower ligand is controlled by metabolic factors rather by the specificity of the phosphoribosyltransferase.

Nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium plays a central role in the synthesis of alpha-ribazole, which is a key component of the lower ligand of cobalamin. Two X-ray structures of CobT are reported here at 1.9 A resolution. First, a complex of CobT with 5,6-dimethylbenzimidazole, and second, a complex of CobT with its reaction products, nicotinate and alpha-ribazole-5'-phosphate. CobT was cocrystallized with 5,6-dimethylbenzimidazole (DMB) in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 A, b = 90.2 A, and c = 47.5 A and one protomer per asymmetric unit. Subsequently, the crystals containing DMB were soaked in nicotinate mononucleotide whereupon the physiological reaction occurred in the crystal lattice to yield nicotinate and alpha-ribazole-5'-phosphate. These studies show that CobT is a dimer where each subunit consists of two domains. The large domain is dominated by a parallel six-stranded beta-sheet with connecting alpha-helices that exhibit the topology of a Rossmann fold. The small domain is made from components of the N- and C-terminal sections of the polypeptide chain and contains a three-helix bundle. The fold of CobT is unrelated to the type I and II phosphoribosylpyrophosphate dependent transferases and does not appear to be related to any other protein whose structure is known. The enzyme active site is located in a large cavity formed by the loops at the C-terminal ends of the beta-strands and the small domain of the neighboring subunit. DMB binds in a hydrophobic pocket created in part by the neighboring small domain. This is consistent with the broad specificity of this enzyme for aromatic substrates [Trzebiatowski, J. R., Escalante-Semerena (1997) J. Biol. Chem. 272, 17662-17667]. The binding site for DMB suggests that Glu317 is the catalytic base required for the reaction. The remainder of the cavity binds the nicotinate and ribose-5'-phosphate moieties, which are nestled within the loops at the ends of the beta-strands. Interestingly, the orientation of the substrate and products are opposite from that expected for a Rossmann fold.

Sweet potato beta-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm x 0.4 mm x 1.0 mm within 2 weeks, belong to the tetragonal space group P4(2)2(1)2 with unit cell dimensions of a = b = 129.63 A and c = 68.42 A. The asymmetric unit contains 1 subunit of beta-amylase, with a crystal volume per protein mass (VM) of 2.57 A3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric beta-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8-2.3 A (with 2 sigma cut-off) with good stereochemistry. The subunit structure of sweet potato beta-amylase (crystallized in the absence of alpha-cyclodextrin) is very similar to that of soybean beta-amylase (complexed with alpha-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent C alpha atoms of the two beta-amylases is 0.96 A. Each subunit of sweet potato beta-amylase is composed of a large (alpha/beta)8 core domain, a small one made up of three long loops [L3 (residues 91-150), L4 (residues 183-258), and L5 (residues 300-327)], and a long C-terminal loop formed by residues 445-493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (alpha/beta)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (alpha/beta)8 barrel.