The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP Cterminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of b-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pulldown assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.

2,3-Cyclic-nucleotide 3-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2,3-cyclic nucleotides to produce 2-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded ␤-sheet and two ␣-helices. The ␤-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2-5 RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.The abundance of the enzyme 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase (CNP 1 ; EC 3.1.4.37) in the central nervous system of all mammals and some other vertebrates such as amphibians and birds has long been an enigma. This derives from the continuing failure to identify a physiological substrate for this enzyme. CNP has an apparent specificity for nucleoside 2Ј,3Ј-cyclic phosphate, which it cleaves to 2Ј-nucleotide end products, none of which (with the exception of NADP/NADPH) are found in metabolite pools. The last 4 decades of research have failed to attribute a function to this protein, although many possibilities have been considered (extensively reviewed in Refs. 1-3). More recently, RICH, a neuronally associated homolog of CNP, has been discovered in fish (4, 5), and the catalytic active site of CNP has been investigated (6).CNP and RICH share catalytic features with three other groups of enzymes: fungal/plant RNA ligases involved in tRNA splicing (7,8), bacterial and archaeal RNA ligases (9) that ligate tRNA half-molecules containing 2Ј,3Ј-cyclic phosphate and 5Ј-hydroxyl termini, and plant and yeast cyclic phosphodiesterases (CPDases) that hydrolyze ADP-ribose 1Љ,2Љ-cyclic phosphate to yield ADP-ribose 1Ј-phosphate (at least one of these latter enzymes also hydrolyzes nucleoside 2Ј,3Ј-cyclic phosphates) (10, 11). These enzymes are thought to play a role in the tRNA-splicing pathways. The x-ray structures of a CPDase from Arabidopsis thaliana (12-14) and, most recently, 2Ј-5Ј RNA ligase from Thermus thermophilus (15) have been determined.Members of this enzyme superfamily occur across a vast range of organisms ranging from bacteria to mammals. It has been suggested (16) that all four classes of enzymes originated from a common ancestor because they all have two similarly spaced histidine-containing tetrapeptides; their catalytic domains have a similar size of ϳ200 residues with similar pattern of predicted secondary structural ...

Rho-dependent transcription termination is an essential process for the regulation of bacterial gene expression. Thus far, only two Rho-specific inhibitors of bacterial transcription termination have been described, the psu protein from the satellite bacteriophage P4 and YaeO from Escherichia coli. Here, we report the solution structure of YaeO, the first of a Rho-specific inhibitor of transcription termination. YaeO is an acidic protein composed of an N-terminal helix and a seven-stranded ␤ sandwich. NMR chemical shift perturbation experiments revealed that YaeO binds proximal to the primary nucleic acid binding site of Rho. Based on the NMR titrations, a docked model of the YaeO-Rho complex was calculated. These results suggest that YaeO binds outside the Rho hexamer, acting as a competitive inhibitor of RNA binding. In vitro gel shift assays confirmed the inhibition of nucleic acid binding to Rho. Site-directed mutagenesis showed that the negative character of YaeO is essential for its function in vivo.

Lipoic acid is an essential prosthetic group in several metabolic pathways. The biosynthetic pathway of protein lipoylation in Escherichia coli involves gene products of the lip operon. YbeD is a conserved bacterial protein located in the dacA-lipB intergenic region. Here, we report the nuclear magnetic resonance structure of YbeD from E. coli. The structure includes a ␤␣␤␤␣␤ fold with two ␣-helices on one side of a four-strand antiparallel ␤-sheet. The ␤2-␤3 loop shows the highest sequence conservation and is likely functionally important. The ␤-sheet surface contains a patch of conserved hydrophobic residues, suggesting a role in protein-protein interactions. YbeD shows striking structural homology to the regulatory domain from D-3-phosphoglycerate dehydrogenase, hinting at a role in the allosteric regulation of lipoic acid biosynthesis or the glycine cleavage system.YbeD is a conserved protein in Escherichia coli located between the dacA gene, which encodes a D-alanyl-D-alanine carboxypeptidase involved in peptidoglycan biosynthesis, and the lip operon, which contains genes that are responsible for lipoic acid biosynthesis.Lipoic acid (1,2-dithiolane-3-pentanoic acid) is a derivative of octanoic acid in which covalently linked sulfur atoms are at the C-6 and C-8 positions. It is an essential prosthetic group used in several metabolic pathways in most living organisms (26). Lipoic acid is covalently attached to the lipoyl domain of certain enzymes via an amide linkage between its carboxylic acid moiety and the ε-amino group of a specific lysine of the lipoylated protein. The added lengths of the reactive lipoate moiety and the lysine side chain create a swinging arm that helps to transfer reaction intermediates between catalytic sites of multienzyme complexes (26). The known E. coli enzymes that use lipoic acid as a prosthetic group include pyruvate and 2-oxoglutarate dehydrogenases (in glycolysis and the citric acid cycle), branched-chain keto acid dehydrogenases (in metabolism of valine, leucine, and isoleucine), and the glycine cleavage system (29, 33). All are involved in oxidative metabolism.There are two complementary pathways of protein lipoylation in E. coli (9). The exogenous pathway utilizes extracellular lipoic acid scavenged from the environment. The enzyme lipoate-protein ligase (LplA) plays a key role in this pathway by using lipoic acid and ATP to lipoylate target proteins (22, 23). The endogenous, biosynthetic pathway is less understood and involves gene products of the lip locus of E. coli. Octanoic acid is a fatty acid precursor of lipoic acid, but its direct conversion to lipoic acid has not been observed (34,35). Instead, the octanoyl moiety on acyl carrier protein of fatty acid synthesis (10) is converted to a lipoyl moiety by the enzyme lipoyl synthase (LipA) (20). This enzyme has 36% sequence identity to biotin synthase (BioB) and is expected to insert sulfur atoms by a similar mechanism (8, 29). The lipoyl moiety is then transferred from acyl carrier protein to the target enzyme by the ...

This paper is concerned with the problem of designing a minimum-cost network of acceptable performance that connects several remote terminals to a central processor using multidrop lines. It is assumed that the message generation rate at each of the 1 terminals is known and that the communication lines of the network have the same capacity. A simple model of the network is used to derive performance constraints for the design procedure. A new heuristic design procedure is proposed. This procedure is compared to other heuristic methods and is found superior in some cases. An improved version of the branch-and-bound (BB) algorithm ofChandy and Russell is developed and tested.