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PubMed2006

Crystal structures of human NUDT5 reveal insights into the structural basis of the substrate specificity.

Human NUDT5 (hNUDT5) is an ADP-ribose pyrophosphatase (ADPRase) belonging to the Nudix hydrolase superfamily. It presumably plays important roles in controlling the intracellular level of ADP-ribose (ADPR) to prevent non-enzymatic ADP-ribosylation by hydrolyzing ADPR to AMP and ribose 5'-phosphate. We report here the crystal structures of hNUDT5 in apo form, in complex with ADPR, and in complex with AMP with bound Mg2+. hNUDT5 forms a homodimer with substantial domain swapping and assumes a structure more similar to Escherichia coli ADPRase ORF209 than human ADPRase NUDT9. The adenine moiety of the substrates is specifically recognized by the enzyme via hydrogen-bonding interactions between N1 and N6 of the base and Glu47 of one subunit, and between N7 of the base and Arg51 of the other subunit, providing the molecular basis for the high selectivity of hNUDT5 for ADP-sugars over other sugar nucleotides. Structural comparisons with E. coli ADPRase ORF209 and ADPXase ORF186 indicate that the existence of an aromatic residue on loop L8 in ORF186 seems to be positively correlated with its enzymatic activity on APnA, whereas hNUDT5 and ORF209 contain no such residue and thus have low or no activities on APnA.

Adenosine Diphosphate Ribose↗
PubMed2005

An all-atom model of the pore-like structure of hexameric VP40 from Ebola: structural insights into the monomer-hexamer transition.

The matrix protein VP40 is an indispensable component of viral assembly and budding by the Ebola virus. VP40 is a monomer in solution, but can fold into hexameric and octameric states, two oligomeric conformations that play central roles in the Ebola viral life cycle. While the X-ray structures of monomeric and octameric VP40 have been determined, the structure of hexameric VP40 has only been solved by three-dimensional electron microscopy (EM) to a resolution of approximately 30 A. In this paper, we present the refinement of the EM reconstruction of truncated hexameric VP40 to approximately 20 A and the construction of an all-atom model (residues 44-212) using the EM model at approximately 20 A and the X-ray structure of monomeric VP40 as templates. The hexamer model suggests that the monomer-hexamer transition involves a conformational change in the N-terminal domain that is not evident during octamerization and therefore, may provide the basis for elucidating the biological function of VP40.

Microscopy, Electron↗
PubMed2004

Structure-activity relationships and structural conformation of a novel urotensin II-related peptide.

Urotensin II (UII) has been described as the most potent vasoconstrictor peptide and recognized as the endogenous ligand of the orphan G protein-coupled receptor GPR14. Recently, a UII-related peptide (URP) has been isolated from the rat brain and its sequence has been established as H-Ala-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. In order to study the structure-function relationships of URP, we have synthesized a series of URP analogs and measured their binding affinity on hGPR14-transfected cells and their contractile activity in a rat aortic ring bioassay. Alanine substitution of each residue of URP significantly reduced the binding affinity and the contractile activity of the peptides, except for the Ala8-substituted analog that retained biological activity. Most importantly, D-scan of URP revealed that [D-Trp4]URP abrogated and [D-Tyr6]URP partially suppressed the UII-evoked contractile response. [Orn5]URP, which had very low agonistic efficacy, was the most potent antagonist in this series. The solution structure of URP has been determined by 1H NMR spectroscopy and molecular dynamics. URP exhibited a single conformation characterized by an inverse gamma-turn comprising residues Trp-Lys-Tyr which plays a crucial role in the biological activity of URP. These pharmacological and structural data should prove useful for the rational design of non-peptide ligands as potential GPR14 agonists and antagonists.

Amino Acid Sequence↗
PubMed2004

The non-structural 3 (NS3) protein of dengue virus type 2 interacts with human nuclear receptor binding protein and is associated with alterations in membrane structure.

Flaviviral infections produce a distinct array of virus-induced intracellular membrane alterations that are associated with the flaviviral replication machinery. Currently, it is still unknown which flaviviral protein(s) is/are responsible for this induction. Using yeast two-hybrid and co-immunoprecipitation analyses, we demonstrated that the NS3 protein of dengue virus type 2 interacted specifically with nuclear receptor binding protein (NRBP), a host cellular protein that influences trafficking between the endoplasmic reticulum (ER) and Golgi, and that interacts with Rac3, a member of the Rho-GTPase family. Co-expression of NS3 and NRBP in baby hamster kidney cells exhibited significant subcellular co-localization, and revealed the redistribution of NRBP from the cytoplasm to the perinuclear region. Furthermore, a set of membrane structures affiliated with the rough ER at the perinuclear region was induced in cells transfected with NS3. These structures are reminiscent of the virus-induced convoluted membranes previously observed in flavivirus-infected cells. This interaction between dengue viral and host cell proteins as well as the formation of the NS3-induced membrane structures suggest that NS3 may subvert the role of NRBP in ER-Golgi trafficking.

Animals↗
PubMed2005

Neuropeptide Y-immunoreactive (NPY-ir) structures in the brain of the gar Lepisosteus oculatus (Lepisosteiformes, Osteichthyes) with special regard to their anatomical relations to gonadotropin-releasing hormone (GnRH)-ir structures in the hypothalamus and the terminal nerve.

The present paper describes neuropeptide Y-like-immunoreactive (NPY-ir) structures in the brain of the spotted gar, Lepisosteus oculatus, with special regard to their anatomical relations to gonadotropin-releasing hormone (GnRH)-ir structures in the hypothalamus and the terminal nerve (TN). NPY-ir cells were found in various locations including the TN, the medial zone of the area dorsalis telencephali, the ventral nucleus of the area ventralis telencephali, the habenula, the dorsal posterior nucleus, the periventricular nucleus of the hypothalamus, the posterior tubercle, the optic tectum, and the lateral part of the tegmentum. NPY-ir fibers were widely distributed throughout the brain except for the cerebellum. They were locally dense in the ventral telencephalon, in the periventricular gray matter of the thalamus and the hypothalamus, and in the ventromedial part of the brainstem, but sparse in the olfactory system. Light-microscopic double immunohistochemistry demonstrated distinct NPY-ir and GnRH-ir structures in the ventral hypothalamus: the NPY-ir system was associated mainly with the periventricular gray matter, whereas the GnRH-ir system was prominent in the external zone of the preoptico-tubero-infundibular area including the median eminence (ME). Here, NPY-ir varicose fibers occasionally abutted on GnRH-ir cells and varicosities or invested GnRH-ir cells, suggesting that NPY directly regulates the function of the hypothalamic GnRHergic neuron system. On the other hand, the TN cells and fibers in the olfactory system were doubly labeled by the antibodies against NPY and GnRH. Immuno-electron-microscopic data strongly suggested that some of the TN fibers projected to the ME.

Animals↗
PubMed1997

Increasing examiner involvement in an objective structured clinical examination by integrating a structured oral examination.

INTRODUCTION: The role of physician examiners in an objective structured clinical examination (OSCE) is relatively passive. In our institution examiners criticized the passive nature of their role. This study evaluates the reliability and viability of adding a structured oral examination to an OSCE. METHOD: Ten 24-minute stations consisted of three parts. Part I: 12 minutes-patient encounter. Part II: 6 minutes-oral presentation covering findings, differential diagnosis, and management plan. Part III: 6 minutes-structural oral examination (SOE), containing 5 predetermined questions. RESULTS: Over 6 consecutive days, 72 graduates were assessed. Overall average score: 72.02 (SD 5.05); reliability 0.84. Part I of the OSCE average score: 69.2 (SD 7.4); reliability 0.69. Part II oral presentation average score 64 (SD 5.8) reliability 0.87. SOE average score 77.7 (SD 6.3); reliability 0.64. Eighty-nine percent of the examiners indicated satisfaction with the new format. CONCLUSIONS: The SOE was a reliable component of an OSCE and contributed to the overall reliability. Examiners reported a higher degree of satisfaction with the examination.

Educational Measurement↗
PubMed1996

Stroke rehabilitation: are highly structured units more conducive to physical activity than less structured units?

OBJECTIVE: To determine if the physical design and organizational structure of rehabilitation units is related to the amount of patients' motor activity. DESIGN: An observational study was conducted; time samples of the motor activity of patients following stroke were taken between 7AM and 7PM both on weekdays and weekends. SETTING: Two rehabilitation units associated with general hospitals with different physical design and organizational structure. One unit was spread over a large area and had a highly organized daily structure; the other was small and informally organized. SUBJECTS: Inpatients with hemiplegia as a result of stroke who gave consent to participate. MAIN OUTCOME MEASURE: The nature and frequency of 14 motor activities were compared between units. RESULTS: No significant difference was found in any of the observed motor activities between the units when using independent groups t tests (p = 0.1-0.8). Subjects in both units spent more than 70% of their day in activities largely unrelated to physical outcome (eg, conversing with visitors or doing nothing observable) and less than 20% of the day in activities that could potentially contribute to their recovery (eg, in therapy or exercising independently). CONCLUSIONS: Rehabilitation units are not functioning as learning environments. The challenge is to identify and implement measures that will change this finding.

Aged↗
PubMed1988

The structure of proteins in aqueous solutions: an assessment of triose phosphate isomerase structure by Fourier-transform infrared spectroscopy.

Recent resolution enhancement and curve-fitting techniques have been applied to infrared spectra from triose phosphate isomerase in aqueous solution, in order to obtain quantitative information on its secondary structure. From our results, 57% alpha-helix, 25% beta-parallel and 10% beta-turns are predicted, in close agreement with the X-ray crystallographic data. On the other hand, measurements of band intensities, both in original and deconvolved spectra are shown to be unreliable for the quantification of secondary structures. The presence of beta-edge structure interacting with the alpha-helical barrel is described and discussed.

Carbohydrate Epimerases↗
PubMed1981

Structural features and the reaction mechanism of cytochrome oxidase: iron and copper X-ray absorption fine structure.

X-ray edge absorption of copper and extended fine structure studies of both copper and iron centers have been made of cytochrome oxidase from beef heart, Paracoccus dentrificans, and HB-8 thermophilic bacteria (1-2.5 mM in heme). The desired redox state (fully oxidized, reduced CO, mixed valence formate and CO) in the x-ray beam was controlled by low temperature (-140 degrees C) and was continuously monitored by simultaneous optical spectroscopy and by electron paramagnetic resonance (EPR) monitoring every 30 min of x-ray exposure. The structure of the active site, a cytochrome a3-copper pair in fully oxidized and in mixed valence formate states where they are spin coupled, contains a sulphur bridge with three ligands 2.60 +/- 0.03 A from Fea3 and 2.18 +/- 0.03 A from Cua3. The distance between Fea3 and Cua3 is 3.75 +/- 0.05 A, making the sulphur bond angle 103 degrees reasonable for sp3 sulphur bonding. The Fea3 first shell has four typical heme nitrogens (2.01 +/- 0.03 A) with a proximal nitrogen at 2.14 +/- 0.03 A. The sixth ligand is the bridging sulphur. The Cua3 first shell is identical to oxidized stellacyanin containing two nitrogens and a bridging sulphur. Upon reduction with CO, the active site is identical to reduced stellacyanin for the Cua3 first shell and contains the sulphur that forms the bridge in fully oxidized and mixed valence formate states. The Fea3 first shell is identical to oxyhemoglobin but has CO instead of O2. The other redox centers, Fea and the other "EPR detectable" Cu are not observed in higher shells of Fea3. Fea has six equidistant nitrogens and Cua has one (or two) nitrogens and three (or two) sulphurs with typical distances; these ligands change only slight on reduction. These structures afford the basis for an oxygen reduction mechanism involving oxy- and peroxy intermediates.

Animals↗
PubMed2002

The crystal structure of the alpha-cellobiose.2 NaI.2 H(2)O complex in the context of related structures and conformational analysis.

The crystal structure of beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranose (alpha-cellobiose) in a complex with water and NaI was determined with Mo K(alpha) radiation at 150 K to R=0.027. The space group is P2(1) and unit cell dimensions are a=9.0188, b=12.2536, c=10.9016 A, beta=97.162 degrees. There are no direct hydrogen bonds among cellobiose molecules, and the usual intramolecular hydrogen bond between O-3 and O-5' is replaced by a bridge involving Na+, O-3, O-5', and O-6'. Both Na+ have sixfold coordination. One I(-) accepts six donor hydroxyl groups and three C-H***I(-) hydrogen bonds. The other accepts three hydroxyls, one Na+, and five C-H***I(-) hydrogen bonds. Linkage torsion angles phi(O-5) and psi(C-5) are -73.6 and -105.3 degrees, respectively (phi(H)=47.1 degrees and psi(H)=14.6 degrees ), probably induced by the Na+ bridge. This conformation is in a separate cluster in phi,psi space from most similar linkages. Both C-6-O-H and C-6'-O-H are gg, while the C-6'-O-H groups from molecules not in the cluster have gt conformations. Hybrid molecular mechanics/quantum mechanics calculations show <1.2 kcal/mol strain for any of the small-molecule structures. Extrapolation of the NaI cellobiose geometry to a cellulose molecule gives a left-handed helix with 2.9 residues per turn. The energy map and small-molecule crystal structures imply that cellulose helices having 2.5 and 3.0 residues per turn are left-handed.

Carbohydrate Conformation↗
PubMed2002

The 1.62 A structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5.

The crystal structure of Thermoascus aurantiacus endoglucanase (Cel5A), a family 5 glycoside hydrolase, has been determined to 1.62 A resolution by multiple isomorphous replacement with anomalous scattering. It is the first report of a structure in the subfamily to which Cel5A belongs. Cel5A consists solely of a catalytic module with compact eight-fold beta/alpha barrel architecture. The length of the tryptophan-rich substrate binding groove suggests the presence of substrate binding subsites -4 to +3. Structural comparison shows that two glycines are completely conserved in the family, in addition to the two catalytic glutamates and six other conserved residues previously identified. Gly 44 in particular is part of a type IV C-terminal helix capping motif, whose disruption is likely to affect the position of an essential conserved arginine. One aromatic residue (Trp 170 in Cel5A), not conserved in term of sequence, is nonetheless spatially conserved in the substrate binding groove. Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite -1.

Ascomycota↗
PubMed1997

Structure of genes for dermaseptins B, antimicrobial peptides from frog skin. Exon 1-encoded prepropeptide is conserved in genes for peptides of highly different structures and activities.

We cloned the genes of two members of the dermaseptin family, broad-spectrum antimicrobial peptides isolated from the skin of the arboreal frog Phyllomedusa bicolor. The dermaseptin gene Drg2 has a 2-exon coding structure interrupted by a small 137-bp intron, wherein exon 1 encoded a 22-residue hydrophobic signal peptide and the first three amino acids of the acidic propiece; exon 2 contained the 18 additional acidic residues of the propiece plus a typical prohormone processing signal Lys-Arg and a 32-residue dermaseptin progenitor sequence. The dermaseptin genes Drg2 and Drg1g2 have conserved sequences at both untranslated ends and in the first and second coding exons. In contrast, Drg1g2 comprises a third coding exon for a short version of the acidic propiece and a second dermaseptin progenitor sequence. Structural conservation between the two genes suggests that Drg1g2 arose recently from an ancestral Drg2-like gene through amplification of part of the second coding exon and 3'-untranslated region. Analysis of the cDNAs coding precursors for several frog skin peptides of highly different structures and activities demonstrates that the signal peptides and part of the acidic propieces are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The organization of the genes that belong to this family, with the signal peptide and the progenitor sequence on separate exons, permits strikingly different peptides to be directed into the secretory pathway. The recruitment of such a homologous 'secretory' exon by otherwise non-homologous genes may have been an early event in the evolution of amphibian.

Amino Acid Sequence↗
PubMed1998

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop.

The synthetic RNA fragment 5'-CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79-97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 A and refined to an R-value of 24.1% also represents the first X-ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.

Base Sequence↗
PubMed2003

Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity.

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class. The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation; in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.

Crystallization↗
PubMed2003

Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA.

Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by </=2-fold. The absence of effects on k(dock), both from atomic modifications and global perturbations, strongly suggests that the transition state for docking is early and does not closely resemble the docked state. These results, the slow rate of docking of 3s(-1), and the observation that a modification that is expected to increase the degrees of freedom between the P1 duplex and the ribozyme core accelerates docking, suggest a model in which a kinetic trap(s) slows docking substantially. Nonetheless, urea does not increase k(dock), suggesting that there is little change in the exposed surface area between the trapped, undocked state and the transition state. The findings highlight that urea and temperature dependencies can be inadequate to diagnose the presence of kinetic traps in a folding process. The results described here, combined with previous work, provide an in-depth view of an RNA tertiary structure formation event and suggest that large, highly structured RNAs may have local regions that are misordered.

Animals↗
PubMed1990

Pattern recognition of the secondary structure of proteins (alpha-helix and beta-structure).

In this paper, an algorithm for the pattern recognition of secondary structure of proteins is proposed. The procedure simultaneously evaluates the contribution of all the residues of a given peptide to its conformation. By means of the algorithm it is possible to select from a universe of well known proteins the most representative alpha-helix and beta-structure peptides, and to use these peptides, as screening matrices to define the unknown structure of any peptide.

Algorithms↗
PubMed1996

The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.

Amino Acid Sequence↗
PubMed2002

Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and catalytic triad.

Kynurenine formamidase (KFase) (EC 3.5.1.9) hydrolyzes N-formyl-L-kynurenine, an obligatory step in the conversion of tryptophan to nicotinic acid. Low KFase activity in chicken embryos, from inhibition by organophosphorus insecticides and their metabolites such as diazoxon, leads to marked developmental abnormalities. While KFase was purportedly isolated previously, the structure and residues important for catalysis and inhibition were not established. KFase was isolated here from mouse liver cytosol by (NH4)2SO4 precipitation and three FPLC steps (resulting in 221-fold increase in specific activity for N-formyl-L-kynurenine hydrolysis) followed by conversion to [3H]diethylphosphoryl-KFase and finally isolation by C4 reverse-phase high-performance liquid chromatography. Determination of tryptic fragment amino acid sequences and cDNA cloning produced a new 305-amino-acid protein sequence. Although an amidase by function, the primary structure of KFase lacks the amidase signature sequence and is more similar to esterases and lipases. Sequence profile analysis indicates KFase is related to the esterase/lipase/thioesterase family containing the conserved active-site serine sequence GXSXG. The alpha/beta-hydrolase fold is suggested for KFase by its primary sequence and predicted secondary conformation. A three-dimensional model based on the structures of homologous carboxylesterase EST2 and brefeldin A esterase implicates Ser162, Asp247 and His279 as the active site triad.

Amino Acid Sequence↗