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DEXD/H-box RNA helicases couple ATP hydrolysis to RNA remodeling by an unknown mechanism. We used x-ray crystallography and biochemical analysis of the human DEXD/H-box protein DDX19 to investigate its regulatory mechanism. The crystal structures of DDX19, in its RNA-bound prehydrolysis and free posthydrolysis state, reveal an alpha-helix that inserts between the conserved domains of the free protein to negatively regulate ATPase activity. This finding was corroborated by biochemical data that confirm an autoregulatory function of the N-terminal region of the protein. This is the first study describing crystal structures of a DEXD/H-box protein in its open and closed cleft conformations.

Impairment of the formation or action of hydrogen sulfide (H(2)S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine beta-synthase and cystathionine gamma-lyase (CSE) are two pyridoxal-5'-phosphate (PLP)-dependent enzymes largely responsible for the production of H(2)S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP.PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H(2)S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE.PLP.PAG complex highlights the particular importance of Tyr(114) in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.

Insulin-like growth factor II (IGF-II) cDNA was isolated from adult guinea pig liver by polymerase chain reaction (PCR) screening. A cDNA sequence was obtained corresponding to part of the preproIGF-II, including the signal peptide, the mature IGF-II and 37 amino acids of the acid carboxy-terminal E-domain. Amino acid sequence prediction, based on the cDNA clone, showed that mature guinea pig IGF-II has a high homology with both human and rat IGF-II, 100 and 94% identity, respectively. Levels of IGF-II mRNA in guinea pigs of different ages were analyzed by solution hybridization/RNase protection assay using part of the isolated IGF-II cDNA as a probe. There is a marked developmental regulation of IGF-II after birth. IGF-II mRNA levels were high in fetal livers, and decreased 15- to 30-fold in adults. As in man, but in contrast to rats, adult guinea pigs have significant levels of IGF-II mRNA in the liver. In fetal guinea pigs, the expression of IGF-II mRNA was 5-, 2- and 70-fold lower in kidney, skeletal muscle and brain cortex, respectively, than in liver. IGF-II mRNA levels in kidney and skeletal muscle of fetal guinea pigs were 5- and 4-fold higher, respectively, compared with adults. Similar sizes of IGF-II mRNA transcripts could be observed on Northern blots in newborn rats and in fetal guinea pigs. Our conclusions are that the mature IGF-II peptide in the guinea pig is 100% identical to the mature peptide in the human. IGF-II mRNA is developmentally regulated in the guinea pig and this developmental regulation of IGF-II expression is different when compared with that of rats, guinea pigs having a higher adult IGF-II expression in liver, kidney and skeletal muscle. Finally, individual IGF-II transcripts may be both differentially and developmentally regulated in guinea pig tissues.