Abstract: DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.
Abstract: Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 A. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Argininosuccinate synthetase catalyzes the transformation of citrulline and aspartate into argininosuccinate and pyrophosphate using the hydrolysis of ATP to AMP and pyrophosphate. This enzymatic process constitutes the rate-limiting step in both the urea and arginine-citrulline cycles. Previous studies have investigated the crystal structures of argininosuccinate synthetase from bacterial species. In this work, the first crystal structure of human argininosuccinate synthetase in complex with the substrates citrulline and aspartate is presented. The human enzyme is compared with structures of argininosuccinate synthetase from bacteria. In addition, the structure also provides new insights into the function of the numerous clinical mutations identified in patients with type I citrullinaemia (also known as classic citrullinaemia).
Abstract: 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.
Abstract: 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.
Abstract: We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.
Abstract: A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.
Abstract: We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.
Abstract: We examined the internal mobility of the estrogen receptor DNA-binding domain (ER DBD) using NMR15N relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied protein fragments consist of residues Arg183-His267 of the human ER and residues Lys438-Gln520 of the rat GR. The15N longitudinal (R1) and transverse (R2) relaxation rates and steady state {1H}-15N nuclear Overhauser enhancements (NOEs) were measured at 30 degrees C at1H NMR frequencies of 500 and 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- to nanosecond time-scales in two regions of the core DBD. These are the extended strand which links the DNA recognition helix to the second zinc domain and the larger loop region of the second zinc domain. The mobility of the corresponding regions of the GR DBD, in particular that of the second zinc domain, is more limited. In addition, we find large differences between the ER and GR DBDs in the extent of conformational exchange mobility on micro- to millisecond time-scales. Based on measurements of R2as a function of the15N refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analysis, we conclude that conformational exchange occurs in the loop of the first zinc domain and throughout most of the second zinc domain of the ER DBD. The conformational exchange dynamics in GR DBD is less extensive and localized to two sites in the second zinc domain. The different dynamical features seen in the two proteins is consistent with previous studies of the free state structures in which the second zinc domain in the ER DBD was concluded to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformational dynamics on the micro- to millisecond time-scales in the free state are involved in intermolecular protein-DNA and protein-protein interactions in the dimeric bound state. Based on the present data and the previously published dynamical and DNA binding properties of a GR DBD triple mutant which recognize an ER binding site on DNA, we argue that the free state dynamical properties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding.
Abstract: Nuclear magnetic resonance was used to compare parameters reflecting solution structure and dynamics of the glucocorticoid receptor DNA-binding domain (GRDBD), which binds specifically to a GRE binding site on DNA, and a triple mutant (GRDBDEGA), which binds to an ERE site. The studies were prompted by an earlier observation that the cooperativity for dimeric DNA-binding is 10 times higher for the GRDBDEGA-ERE association than for the GRDBD-GRE association (Lundbäck et al., 1994). The higher binding cooperativity of the mutant was unexpected since the triple mutation (G458E, S459G, and V462A) is made in the recognition helix and distant from the dimerization surface which is formed by residues in the fragment A477-N491. Sequential and long-range NOE connectivities and measured 3JHNHalpha coupling constants indicate that the overall structures of the two proteins are very similar, possibly with a less well-defined structure of the fragment K486-N491 in GRDBDEGA. However, chemical shift changes, line broadening, and increased amide proton exchange rates are observed for several residues at, or close to, the dimerization surface of the mutant. These observations are interpreted as a lower stability and/or several slowly interconverting folded conformations of this region of GRDBDEGA. The effects are likely to be due to the loss of a hydrogen bond which links S459 to the dimerization region in GRDBD. Different mechanisms for the increased binding cooperativity of the mutant are discussed, and it is noted that the properties of the GRDBDEGA dimerization region are reminiscent of those reported for the estrogen receptor DBD, which also binds to an ERE site.
Abstract: 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: High-performance liquid chromatography based on pre-column derivatization of histidine and 3-methylhistidine with o-phthalaldehyde-mercaptoethanol has been used to determine these amino acids in small volumes of plasma and urine. The elution is performed in 45 min on a 5-micron Resolve C18 column by a multi-step gradient. The eluted analytes are measured with a fluorescence detector which provides detection limits of less than 1 pmol per 20-microliter injection. The correlation between concentration and the integrated area of amino acids gives a linear relationship between 10 and 150 pmol per injection. Some preliminary results from patients with chronic renal failure under variation of diet are presented.
