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Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740-748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.

Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.

The endoplasmic reticulum (ER) stress response detects malfunctions in cellular physiology, and microbial pattern recognition receptors recognize external threats posed by infectious agents. This study has investigated whether proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress. Activation of ER stress, in combination with Toll-like receptor (TLR) agonists, markedly enhanced expression of mRNA of the unique p19 subunit of IL-23, and also significantly augmented secretion of IL-23 protein. These effects were not seen for IL-12 secretion. The IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation. Knockdown of CHOP in U937 cells significantly reduced the synergistic effects of TLR and ER stress on IL-23p19 expression, but did not affect expression of other LPS-responsive genes. The integration of ER stress signals and the requirement for CHOP in the induction of IL-23 responses was also investigated in a physiological setting: infection of myeloid cells with Chlamydia trachomatis resulted in the expression of CHOP mRNA and induced the binding of CHOP to the IL-23 promoter. Furthermore, knockdown of CHOP significantly reduced the expression of IL-23 in response to this intracellular bacterium. Therefore, the effects of pathogens and other environmental factors on ER stress can profoundly affect the nature of innate and adaptive immune responses.

Objective: Single nucleotide polymorphisms (SNPs) in intron 1 of fat mass- and obesity-associated gene (FTO) are strongly associated with human adiposity, whereas Fto(-/-) mice are lean and Fto(+/-) mice are resistant to diet-induced obesity. We aimed to determine whether FTO mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure-function relationships within FTO., Research Design and Methods: We sequenced all coding exons of FTO in 1,433 severely obese and 1,433 lean individuals. We studied the enzymatic activity of selected nonsynonymous variants., Results: We identified 33 heterozygous nonsynonymous variants in lean (2.3%) and 35 in obese (2.4%) individuals, with 8 mutations unique to the obese and 11 unique to the lean. Two novel mutations replace absolutely conserved residues: R322Q in the catalytic domain and R96H in the predicted substrate recognition lid. R322Q was unable to catalyze the conversion of 2-oxoglutarate to succinate in the presence or absence of 3-methylthymidine. R96H retained some basal activity, which was not enhanced by 3-methylthymidine. However, both were found in lean and obese individuals., Conclusions: Heterozygous, loss-of-function mutations in FTO exist but are found in both lean and obese subjects. Although intron 1 SNPs are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that FTO has a role in energy balance, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese. Functional analyses of FTO mutations have given novel insights into structure-function relationships in this enzyme.

Lipodystrophic syndromes are characterized by adipose tissue deficiency. Although rare, they are of considerable interest as they, like obesity, typically lead to ectopic lipid accumulation, dyslipidaemia and insulin resistant diabetes. In this paper we describe a female patient with partial lipodystrophy (affecting limb, femorogluteal and subcutaneous abdominal fat), white adipocytes with multiloculated lipid droplets and insulin-resistant diabetes, who was found to be homozygous for a premature truncation mutation in the lipid droplet protein cell death-inducing Dffa-like effector C (CIDEC) (E186X). The truncation disrupts the highly conserved CIDE-C domain and the mutant protein is mistargeted and fails to increase the lipid droplet size in transfected cells. In mice, Cidec deficiency also reduces fat mass and induces the formation of white adipocytes with multilocular lipid droplets, but in contrast to our patient, Cidec null mice are protected against diet-induced obesity and insulin resistance. In addition to describing a novel autosomal recessive form of familial partial lipodystrophy, these observations also suggest that CIDEC is required for unilocular lipid droplet formation and optimal energy storage in human fat.

FTO is a nuclear protein belonging to the AlkB-related non-haem iron- and 2-oxoglutarate-dependent dioxygenase family. Although polymorphisms within the first intron of the FTO gene have been associated with obesity, the physiological role of FTO remains unknown. Here we show that a R316Q mutation, inactivating FTO enzymatic activity, is responsible for an autosomal-recessive lethal syndrome. Cultured skin fibroblasts from affected subjects showed impaired proliferation and accelerated senescence. These findings indicate that FTO is essential for normal development of the central nervous and cardiovascular systems in human and establish that a mutation in a human member of the AlkB-related dioxygenase family results in a severe polymalformation syndrome.

Acute intermittent porphyria is an autosomal dominantly inherited disorder, classified as acute hepatic porphyria, caused by a deficiency of hydroxymethylbilane synthase (EC 2.5.1.61, EC 4.3.1.8, also known as porphobilinogen deaminase, uroporphyrinogen I synthase), the third enzyme in heme biosynthesis. Clinical features include autonomous, central, motor or sensory symptoms, but the most common clinical presentation is abdominal pain caused by neurovisceral crises. A diagnosis of acute intermittent porphyria is crucial to prevent life-threatening acute attacks. Detection of DNA variations by molecular techniques allows a diagnosis of acute intermittent porphyria in situations where the measurement of porphyrins and precursors in urine and faeces and erythrocyte hydroxymethylbilane synthase activity is inconclusive. In the present study, we identified gene defects in six Czech patients with acute intermittent porphyria, as diagnosed based on biochemical findings, and members of their families to confirm the diagnosis at the molecular level and/or to provide genetic counselling. Molecular analyses of the hydroxymethylbilane synthase gene revealed seven mutations. Four were previously reported: c.76C>T, c.77G>A, c.518G>A, c.771 + 1G>T (p.Arg26Cys, p.Arg26His, p.Arg173Gln). Three were novel mutations: c.610C>A, c.675delA, c.750A>T (p.Gln204Lys, p.Ala226ProfsX28, p.Glu250Asp). Of particular interest, one patient had two mutations (c.518G>A; c.610C>A), both located in exon 10 of the same allele. To establish the effects of the mutations on enzyme function, biochemical characterization of the expressed normal recombinant and mutated proteins was performed. Prokaryotic expression of the mutant alleles of the hydroxymethylbilane synthase gene revealed that, with the exception of the p.Gln204Lys mutation, all mutations resulted in little, if any, enzymatic activity. Moreover, the 3D structure of the Escherichia coli and human protein was used to interpret structure-function relationships for the mutations in the human isoform.

Mutations in the hydroxymethylbilane synthase (HMBS) gene are responsible for the inherited disorder of acute intermittent porphyria (AIP). AIP is diagnosed on the basis of characteristic clinical symptoms, elevated levels of urinary porphyrin precursors aminolevulinic acid (ALA) and porphobilinogen (PBG) and a decreased erythrocytic HMBS activity, although an identifiable HMBS mutation provides the ultimate proof for AIP. Six Israeli AIP families underwent biochemical and mutation analysis in order to establish an AIP diagnosis. Variability with respect to the ALA/PBG levels and HBMS activity was found among the index patients. Indeed, each family carried a unique mutation in the HMBS gene. A novel missense c.95G>C (p.R32P) was shown to be a de novo mutation in one family, along with five known mutations p.T59I, p.D178N, p.V215M, c.730&#95;731delCT and c.982&#95;983delCA identified in the rest of the families. Both R32P and D178N were expressed in a prokaryotic system. Recombinant p.R32P was enzymatically inactive as demonstrated by a <1% residual activity, whereas p.D178N possessed 81% of the activity of the wild type enzyme. However, the p.D178N mutant did display a shift in optimal pH and was thermo labile compared to the wild type. Among the four missense mutations, p.R32P and p.V215M had not only harmful effects on the enzyme in vitro but also were associated with high levels of ALA/PBG in patients. On the other hand, the in vitro effect of both p.T59I and p.D178N, and the impact of these mutations on the enzyme structure and function as interpreted by the 3-D structure of the Escherichia coli enzyme, were weaker than that of p.R32P and p.V215M. Concomitantly, patients carrying the p.T59I or p.D178N had normal or borderline increases in ALA/PBG concentrations although they presented characteristic clinical symptoms. These findings provided further insights into the causal relationship between HMBS mutations and AIP.

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.

Based on Internet search, we were contacted by a 50-year-old man suffering from severe abdominal pain. Acute hepatic porphyria was considered from positive Watson-Schwartz test. He, not being a health professional, searched for centres with ability to do molecular diagnosis and for information about therapeutic possibilities. He asked his physician for haem-arginate (Normosang, Orphan Europe, Paris) treatment, arranged sending his blood to our laboratory and mediated genetic counselling for him and his family. Molecular analyses of the PBGD gene revealed a novel mutation in exon 15, the 973insG. Subsequently, genetic analysis was performed in 18 members of the proband's extensive family. In 12 members of the family, the same mutation was found. The mutation, which consisted of one nucleotide insertion, resulted in addition of four different amino acids leading to a protein that is prematurely truncated by the stop codon. The effect of this mutation was investigated by expression of the wildtype and mutated PBGD in a prokaryotic expression system. The mutation resulted in instability of the protein and loss of enzymatic function. The increasing access to a number of disease- and symptom-oriented web pages presents a new and unusual venue for gaining knowledge and enabling self-diagnosis and self-help. It is, therefore, important that diseaseoriented Internet pages for public use should be designed with clarity and accurate current knowledge based background.

bir-1, a Caenorhabditis elegans inhibitor-of-apoptosis gene homologous to Survivin is organized in an operon with the transcription cofactor C. elegans SKIP (skp-1). Because genes arranged in operons are frequently linked functionally, we have asked whether BIR-1 also functions in transcription. bir-1 inhibition resulted in multiple developmental defects that overlapped with C. elegans SKIP loss-of-function phenotypes: retention of eggs, dumpy, movement defects, and lethality. bir-1 RNA-mediated interference decreased expression of several gfp transgenes and the endogenous genes dpy-7 and hlh-1. Immunoblot analysis revealed decreased phosphoacetylated histones in bir-1 RNA-mediated interference-treated worms. In a heterologous transfection system, BIR-1 augments thyroid hormone-regulated transcription and has an additive effect with SKIP. These results show that BIR-1 functions in the regulation of transcription and development.

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).

An infant with Donohue's syndrome (leprechaunism) was found to be homozygous for an in-frame trinucleotide deletion within the insulin receptor gene resulting in the deletion of valine 335. When transiently transfected into Chinese hamster ovary cells, mutant receptor was produced in a mature form, but at significantly lower levels compared with wild-type receptor. Cell surface biotinylation experiments revealed that significant amounts of the DeltaV335 receptor were expressed on the cell surface. Despite this, cells expressing this receptor showed no significant insulin binding or ligand-induced receptor autophosphorylation. Although the DeltaV335 receptor was capable of being immunoprecipitated with antibodies directed against the beta-subunit of the receptor, the mutant receptor could not be recognized by a panel of antibodies directed against different epitopes of the alpha-subunit, suggesting that the loss of V335 results in a major conformational alteration in the receptor alpha-subunit. This would be predicted by the positioning of V335 at a critical location within a strand that provides the main rigid scaffold for the two beta-sheet faces of the L2 domain of the receptor. The severe biochemical and clinical consequences of this novel mutation, which occur despite substantial expression on the cell surface, emphasize the crucial role of the L2 domain in ligand binding by the insulin receptor.

There has recently been considerable interest in using NMR spectroscopy to identify ligand binding sites of macromolecules. In particular, a modular approach has been put forward by Fesik et al. (Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531-1534) in which small ligands that bind to a particular target are identified in a first round of screening and subsequently linked together to form ligands of higher affinity. Similar strategies have also been proposed for in silico drug design, where the binding sites of small chemical groups are identified, and complete ligands are subsequently assembled from different groups that have favorable interactions with the macromolecular target. In this paper, we compare experimental and computational results on a selected target (FKBP12). The binding sites of three small ligands ((2S)1-acetylprolinemethylester, 1-formylpiperidine, 1-piperidinecarboxamide) in FKBP12 were identified independently by NMR and by computational methods. The subsequent comparison of the experimental and computational data showed that the computational method identified and ranked favorably ligand positions that satisfy the experimental NOE constraints.

SKI-binding protein (SKIP) is a transcription cofactor present in all eukaryotes. Here we show that SKIP is a unique protein that is required for Caenorhabditis elegans viability and development. Expression of CeSKIP (skp-1) assayed by RT-PCR and by GFP fluorescence in transgenic lines starts in embryos and continues to adulthood. Loss of CeSKIP activity by RNA-mediated inhibition results in early embryonic arrest similar to that seen following inhibition of RNA polymerase II. RNA polymerase II phosphorylation appears normal early in CeSKIP RNA-mediated inhibition treated embryos although the expression of several embryonic GFP reporter genes is severely restricted or absent. Our data suggest that CeSKIP is an essential component of many RNA polymerase II transcription complexes and is indispensable for C. elegans development.

Assignment of the resonances in nuclear magnetic resonance spectra is considered a pre-requisite for the interpretation of spectra that yield structural information. The determination of the three-dimensional structure of a biological macromolecule may, however, be achieved directly without spectral assignment, using the same set of heteronuclear scalar and dipolar coupling experiments as normally used. A cross-peak in any of the spectra may be interpreted as a distance between atoms, yielding a set of distances between unassigned atoms that serves to define the tertiary structure of the molecule. The principle is illustrated using the 76 amino acid protein ubiquitin.

The structure of a recently reported neurotrophic ligand, 3-(3-pyridyl)-1-propyl(2S)-1-(3,3-dimethyl-1, 2-dioxopentyl)-2-pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in a trans conformation. However, fewer favourable protein-ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506, 15N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent-exposed residues which are important for mediating the interaction of immunophilin/ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.

The interaction of Li+ with myo-inositol monophosphatase was studied by 7Li-NMR spectroscopy. Li+ binding to the enzyme induces a downfield shift and broadening of the 7Li-NMR signal. Changes of the chemical shift were used to follow the titration of the enzyme with lithium and to determine a dissociation constant, Kd = (1.0 +/- 0.1) mM. Only one major binding site/enzyme subunit was inferred. The complex forms independently of the presence of inorganic phosphate. Metals from the group IIa of the periodic table compete with Li+ binding with the affinity increasing in the order Mg2+ < Ca2+ < Be2+. In contrast to lithium, their binding is enhanced by phosphate.

The structure of the disintegrin echistatin has been determined by 1H NMR, distance geometry calculations and restrained molecular dynamics simulations. The structure has been refined from the preliminary distance geometry calculations with the inclusion of additional 1H NMR data and hydrogen bonds identified in early stages of the molecular dynamics calculations. The calculations reported here allow a distinction to be made between the two possible disulfide bridging patterns-echistatin is crosslinked as follows: Cys2-Cys11, Cys7-Cys32, Cys8-Cys37, Cys20-Cys39. The final set of structures gives an average pairwise root mean square distance of 0.100 nm (calculated over the backbone atoms of residues Ser4-Cys20 and Asp30-Pro40). The core of echistatin is a well defined though irregular structure, composed of a series of non-classical turns crosslinked by the disulfide bridges and stabilised by hydrogen bonds. The RGD sequence is located in a protruding loop whose stem is formed by two rigid, hydrogen-bonded strands (Thr18-Cys20, Asp30-Cys32). The RGD sequence is connected to this structure by short, flexible segments. High (but not unlimited) mobility is probably necessary for fast recognition and fitting to the integrin receptors. Sequence variability among the disintegrins is found in the segments flanking the RGD sequence, suggesting that these may be important in conferring specificity for the receptors.

The structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase I alpha (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the protein/peptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp-2 and Phep-11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate Arg-Lys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate. Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80 degrees C with no clear melting transition. Assignment of the resonances in the 1H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.(ABSTRACT TRUNCATED AT 400 WORDS)

Integrin binding to proteins often involves recognition of domains containing the arginine-glycine-aspartate (RGD) motif. Different binding affinities and specificities of the integrin-ligand protein interactions involve additional protein domains. The n.m.r. structure of the snake-venom protein echistatin suggested that the C-terminal portion of the molecule might be important, in addition to the RGD domain, in binding to the integrin glycoprotein IIbIIIa (GPIIbIIIa) [Saudek, Atkinson and Pelton (1991) Biochem. 30, 7369-7372]. The synthetic C-terminal peptide, echistatin-(40-49), PRNPHKGPAT, (1) inhibited binding of GPIIbIIIa to immobilized echistatin (IC50 3-6 mM), but did not inhibit binding of GPIIbIIIa to immobilized fibrinogen (up to 5 mM peptide), (2) activated GPIIbIIIa binding to fibronectin and vitronectin, usual ligands for the activated integrin, (3) activated binding of GPIIbIIIa to collagen type I and type IV, proteins not usually regarded as ligands for the integrin, and (4) stimulated 125I-fibrinogen binding by human platelets. These findings argue for an interaction of this non-RGD domain in echistatin with GPIIbIIIa, leading to activation of the integrin and extension of the ligand specificity to include immobilized collagen.

A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GrAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180 degrees radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2 mM solution of a double-stranded DNA fragment in 90% H2O at 5 degrees C is presented.

The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D2O was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11-36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N-terminus (residues 1-9) appears as an unstructured mobile segment. Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 +/- 0.6 microM at pH 2-8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.

Cyclic GMP dependent protein kinase exists as a dimer in its native form. A peptide corresponding to the dimerization domain in the N-terminal segment has been characterized by circular dichroism, ultracentrifugation, and 1H NMR spectroscopy. The peptide (G-kinase1-39 amide) is shown to be dimeric in solution. Determination of the molecular weight of the species in solution from the sedimentation coefficient and diffusion coefficient yields a value more than twice that of the monomeric species. Circular dichroism studies show G-kinase1-39 amide to be largely helical in aqueous solution and stable over a wide range of pH and temperature. The conformational stability is found to be concentration dependent, the peptide having a melting temperature of 75 degrees C (at 20 microM and pH 4.0). The assignment of the 1H NMR spectrum and analysis of the patterns of nuclear Overhauser enhancements confirm the helical nature of the conformation. Distance geometry calculations result in a well-defined helical structure containing a kink near Ser 26. The dimerization of G-kinase is most likely to occur through the hydrophobic interaction of leucine and isoleucine side chains located on one face of a helical structure with supporting electrostatic interactions between flanking side chains. The dimerization domain of G-kinase is clearly analogous to the "leucine zipper" motifs found in a number of DNA transcriptional activators.

Echistatin is a 49 amino acid protein isolated from the venom of a viper (Echis carinatus) and is one of the smallest natural adhesive ligands that interacts with integrin-type receptors through an Arg-Gly-Asp (RGD) sequence. The structure of echistatin in aqueous solution has been determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 490 interatomic distance constraints, which were used in distance geometry calculations. The chain is shown to fold in a series of irregular loops to form a rigid core stabilized by four cystine cross-links. From this core an irregular hairpin and the C-terminus protrude. The core and the hairpin are further stabilized by a network of hydrogen bonds. The RGD sequence is located in a mobile loop at the tip of the hairpin. The mobility and its significance for activity are discussed.

We report the complete structure determination of a 34 residue synthetic peptide with the amino acid sequence of the dimerization domain (leucine zipper) of GCN4. A high resolution structure in solution was obtained by 1H-NMR studies and distance geometry calculations followed by restrained energy minimization. A set of 20 final structures was obtained with an average root mean square deviation of 1.3 A for the backbone atoms (excluding the first and the last two residues). The structure contains an uninterrupted helix. A comparison with a structure previously determined for a larger peptide containing both the DNA-binding region (basic region) and the leucine-zipper motif shows the structural independence of the leucine-zipper domain from the contiguous DNA binding region.

The solution conformation of Endothelin-1, a recently discovered bicyclic, 21 amino acid peptide, has been examined by 1H NMR in deuterated dimethylsulphoxide and circular dichroism in aqueous and organic solvents. A total of 158 NOEs were detected, which were used as distance constraints in the distance geometry program DISGEO. Two families of structures were obtained, both characterized by a helix-like region extending from Lys9 to Cys15, but with opposite "handedness". Circular dichroism studies of the peptide in both aqueous and trifluoroethanol solutions show a negative shoulder at 224 nm, characteristic of right-handed helices. Molecular dynamics and energy minimization yielded a solution structure for this new peptide compatible with all experimental observations.

The structure of the basic region (i.e., the region responsible for sequence-specific binding to DNA) of the transcriptional activator GCN4 was studied. Two peptide fragments containing either the basic region alone (residues 240-280) or the basic and the dimerization leucine zipper domains (220-280) were synthesized and investigated by nuclear magnetic resonance and circular dichroic spectroscopy. The basic region in the absence of DNA appears as a mobile flexible segment folded into a loose helix. The helical stability increases upon addition of trifluoroethanol and/or lowering of the temperature. Dimerization via the leucine zipper does not affect the three-dimensional structure of the basic region. Possible consequences for the binding to DNA are discussed.

Salmon calcitonin, a 32-residue peptide with a 1-7 disulfide bridge, was synthesized by standard solid-phase techniques, and studied by CD and two-dimensional NMR experiments. The peptide was dissolved in pure trifluoroethanol (TFE) and in aqueous solutions containing various amounts of TFE. CD studies in pure TFE indicated the presence of an alpha-helical structure comprising 40% of the constituent amino acids. This was fully confirmed by nmr. A detailed analysis was performed with the peptide in a 9 : 1 deuterated TFE/H2O mixture. A total of 365 nuclear Overhauser enhancements (154 intraresidual, 112 sequential and 99 long range) were complied from the nuclear Overhauser enhancement spectroscopy spectra and used in the distance geometry calculations. The core of the peptide between residues 8 and 22 assumes an alpha-helix like structure. The Cys 1-Cys 7 ring is well defined and in close association with the helix, while the C-terminal decapeptide folds back toward the core, forming a loose loop.

Endothelin-3 dissolved in 10% aqueous acetic acid was studied by nuclear magnetic resonance spectroscopy. A total of 363 distances (143 intra-residue, 108 sequential and 112 long range) was compiled from the nuclear Overhauser effect spectra and used in distance geometry calculations. The molecule assumes a compact conformation stabilized by hydrophobic interactions of the side chains. There is a helix-like structure between the residues 9-15 and an extended strand at the N-terminus. The C-terminus is in close proximity to the bicyclic ring.

When calculating three-dimensional structures from NMR data, alternative solutions with very large RMS deviation can be obtained. Sometimes local or global inversions of the protein folding can be observed. We call these different solutions topological mirror images, as they keep the correct amino acid chirality. They are observed when the number of restraints is insufficient and represent different solutions from the same scalar information. Therefore they are common in small peptides where the NMR data are often limited and the secondary structure is not very well defined. They can also be observed in large molecules in regions of higher flexibility. In our experience the observation of topological mirror images is independent of the efficiency of sampling of the algorithm used. We present four examples of proteins with different size and folding. We also discuss ways to distinguish among the different solutions.

The solution structure of an active synthetic peptide containing both the leucine zipper and the adjacent basic domain of the yeast transcription factor GCN4 (residues 220-280) was determined by NMR. The two domains show structurally distinct behaviours. In the absence of DNA, the basic domain is, although very flexible, structured and fluctuating around a helical conformation. The leucine zipper region forms a long, uninterrupted helix. From a suitable set of NMR distances the three-dimensional structure of the leucine zipper monomeric sub-domain was calculated by distance geometry algorithms. The structure of the symmetrical parallel dimer was obtained by model building using the NMR information. A smaller peptide with the sequence of the isolated basic region (residues 1-35 of the 61 residue peptide) was also synthesized. Circular dichroism studies showed 30-40% helicity. A flexible helix spans the region between residues 8 and 21. The comparison of our results with suggested models is discussed in detail.

Sequence-specific assignment of the 1H NMR spectrum of the 36 amino acid polypeptide porcine neuropeptide Y (pNPY) at pH 3.1 is reported. It was achieved by use of standard two-dimensional techniques and by a combination of the sequential and main-chain-directed assignment strategies. The secondary structure was derived from inspection of the nuclear Overhauser spectra, slow hydrogen-deuterium exchange effects, chemical shifts of main-chain HA resonances, and coupling constants. These studies indicate that the C-terminal segment (residues 11-36) folds into an amphiphilic alpha-helix; the N-terminal segment, containing three prolines in both cis and trans conformations, assumes no regular structure. CD studies of pNPY at pH 3.1 and 7.4 show an increase in ordered structure at neutral pH. The difference spectrum, however, is typical of an alpha-helix and suggests a stabilization of residues 11-36, possibly via Maxfield-Scheraga pair interactions involving side-chain residues. This is supported by a comparison of the one-dimensional 1H NMR spectra of pNPY at pH 3.1 and 7.4, where no remarkable differences are observed.

The antigenic properties of acylphosphatase are compared with its various sequential characteristics (hydrophobicity, chemical shift of the main-chain 1H-NMR resonances, numbers and intensities of the nuclear Overhauser enhancements, hydrogen-deuterium exchange and sequential arrangement of the secondary structure units). The discussion is based on the complete sequential assignment of the 1H-NMR spectrum and the knowledge of the three-dimensional fold of the protein obtained by NMR spectroscopy from distance geometry calculations. Regions with very different degrees of mobility can be distinguished. It is found that all major antigenic sites are located in the most mobile surface loops.

Three derivatives of poly[alpha-N beta-(2-hydroxyethyl)-DL-asparagine, beta-N alpha-(2-hydroxyethyl)-DL-asparagine] were used as soluble carrier polymers for binding the trypsin-kallikrein inhibitor (TKI). In addition to the parent polymer an anionic derivative with 19.2 mol of carboxyl groups/100 mol of residues and a hydrophobized derivative with 18.0 mol of butyl groups/100 mol of residues were also used. All carriers contained 6.6 mol of 2-(4-aminobenzamido)ethyl residues/100 mol which were used for the binding after diazotation. All derivatives retained a high inhibitory activity toward trypsin and chymotrypsin whereas the effect on kallikrein was substantially reduced. The kinetic constants were estimated under the simplifying assumption, that classical kinetic equations for the enzyme-inhibitor interaction may be applied. The equilibrium dissociation constants (5 X 10(-9) mol/l) and association rate constants (1 X 10(5) l X mol-1 X s-1) with respect to trypsin are unaffected by the two chemical modifications of the carrier. The increase in the equilibrium dissociation constant (compared to native TKI) is probably a result of the decreasing strength of the enzyme-inhibitor complex rather than a manifestation of reduced rate of diffusion of the enzyme to the active center of the polymer-bound inhibitor.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.

Various His-Pt(II) coordination compounds were prepared by reaction of K2PtCl4 or cis-[Pt(NH3)2Cl2](cis-DDP) with His and analyzed by 1H and 13C NMR spectroscopy, electrophoresis, and ion-exchange chromatography. His may be coordinated to Pt by the imidazol iminogroup and/or the alpha-aminogroup; the carboxygroup remains always free. Both bidentate as well as monodentate ligands were identified. Cis-DDP reacts with His to give a mixture of compounds where all these possibilities are present: cis-diamine-(histidine-N,N-)Pt(II) and three different types of cis-diammine-bis(histidine). HCl trans cleavage of compounds with bidentate His ligands leads to a mixture of two compounds having His ligated to Pt by an amino or imin group. The methods applied are suitable for analyzing reactions of His with cis-DDP under model conditions similar to physiological conditions.

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.

Various Pt(II)-glycine coordination compounds were characterized by 1H and 13C NMR spectroscopy, some of them also by electrophoretic and chromatographic behavior. The results were applied to the analysis of the reaction mixtures of cis-[Pt(NH3)2Cl2] and glycine obtained under various conditions. Cis-[Pt(NH3)2Cl2] reacts with glycine to give cis-diammine-(glycine-N,O)-Pt(II) and cis-diammine-bis(glycine-N-)Pt(II). Their ratio depends primarily on the pH of the reaction medium. Conformation of these compounds is discussed based on the observed Pt-C and Pt-H NMR coupling constants.

The chromotest which measures the induction of SOS functions in an Escherichia coli strain bearing the lacZ gene under the sfiA gene control was evaluated as a screening method for platinum complexes with antitumor activity. Four types of complexes were used: (a) those with two nitrogen ligands in cis position and chlorine or carboxylic acid as anionic ligand; (b) four nitrogen atoms surrounding platinum; (c) complexes involving sulphur and amino group of methionine; (d) complexes of tetravalent platinum. The trans-diaminodichloro complex was used as negative control. Groups (b) and (c) were inactive in the chromotest which correlated well with their absence of antitumor activity. Antitumor activity and a positive chromotest also correlated in group (d). In group (a), which includes complexes with well established antitumor activity, the chromotest separated complexes with fast and slow rate of hydrolysis. The results fitted well in the mutation induction test.

A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the identification of the main-chain alpha and amide proton resonances. Several of the completely assigned spin systems were then identified as being part of the secondary structure units which led, after analysis of the primary amino acid sequence, to unambiguous sequence-specific assignments. The identification and assignment of the remaining side-chain resonances was then completed and are reported here. These results provide a complete data base for the three-dimensional structure determination of this enzyme in solution.

It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to locate the two segments within the primary structure (residues 22 to 35 and 55 to 66), thus providing the sequential assignment. The assignment of the side-chains was completed and a list of all nuclear magnetic resonance constraints within the two segments (126 intra- and 180 interresidue distances, 21 torsion angles phi and 19 hydrogen bonds) was produced. Distance geometry calculation shows that each segment forms an alpha-helix. The mutual orientation of the two helices was established subsequently.

Sequence-specific assignment of 1H nuclear magnetic resonance spectra of acylphosphatase (EC 3.6.1.7) isolated from rabbit skeletal muscle have made it possible to identify short distance constraints from nuclear Overhauser enhancement spectra, to evaluate spin-spin coupling constants of many backbone amide hydrogens and to assess their slow exchange with deuterons in 2H2O solution. Analysis of these data show that the major regular secondary structure of the enzyme consists of five extended beta-strands, four of which are arranged in an antiparallel beta-sheet, while the fifth is attached parallel. A helix consisting of 11 residues has also been identified. Consideration of additional distance constraints between sequentially remote residues has allowed us to give an outline of the overall fold of the protein.

The solution conformation of the recently discovered bi-cyclic, 21 amino acid vasoconstrictor peptide, Endothelin I, has been examined by 1H-NMR in deuterated dimethyl sulphoxide. A full sequential assignment has been achieved. In addition, 19 long range NOEs were detected which were employed as distance constraints in molecular dynamics calculations to yield a possible solution structure for this new peptide.