NMR spectroscopic analysis of the first two steps of the pentose-phosphate pathway elucidates the role of 6-phosphogluconolactonase.

The pentose-phosphate pathway provides reductive power and nucleotide precursors to the cell through oxidative and nonoxidative branches, respectively. 6-Phosphogluconolactonase is the second enzyme of the oxidative branch and catalyzes the hydrolysis of 6-phosphogluconolactones, the products of glucose 6-phosphate oxidation by glucose-6-phosphate dehydrogenase. The role of 6-phosphogluconolactonase was still questionable, because 6-phosphogluconolactones were believed to undergo rapid spontaneous hydrolysis. In this work, nuclear magnetic resonance spectroscopy was used to characterize the chemical scheme and kinetic features of the oxidative branch. We show that 6-phosphogluconolactones have in fact a nonnegligible lifetime and are highly electrophilic compounds. The delta form (1-5) of the lactone is the only product of glucose 6-phosphate oxidation. Subsequently, it leads to the gamma form (1-4) by intramolecular rearrangement. However, only the delta form undergoes spontaneous hydrolysis, the gamma form being a "dead end" of this branch. The delta form is the only substrate for 6-phosphogluconolactonase. Therefore, 6-phosphogluconolactonase activity accelerates hydrolysis of the delta form, thus preventing its conversion into the gamma form. Furthermore, 6-phosphogluconolactonase guards against the accumulation of delta-6-phosphogluconolactone, which may be toxic through its reaction with endogenous cellular nucleophiles. Finally, the difference between activity of human, Trypanosoma brucei, and Plasmodium falciparum 6-phosphogluconolactonases is reported and discussed.

Role of the substrate conformation and of the S1 protein in the cleavage efficiency of the T4 endoribonuclease RegB.

The T4 endoribonuclease RegB is involved in the inactivation of the phage early messengers. It cuts specifically in the middle of GGAG sequences found in early messenger intergenic regions but not GGAG sequences located in coding sequences or in late messengers. In vitro RegB activity is very low but is enhanced by a factor up to 100 by the ribosomal protein S1. In the absence of clear sequence motif distinguishing substrate and non-substrate GGAG-containing RNAs, we postulated the existence of a structural determinant. To test this hypothesis, we correlated the structure, probed by NMR spectroscopy, with the cleavage propensity of short RNA molecules derived from an artificial substrate. A kinetic analysis of the cleavage was performed in the presence and absence of S1. In the absence of S1, RegB efficiently hydrolyses substrates in which the last G of the GGAG motif is located in a short stem between two loops. Both strengthening and weakening of this structure strongly decrease the cleavage rate, indicating that this structure constitutes a positive cleavage determinant. Based on our results and those of others, we speculate that S1 favors the formation of the structure recognized by RegB and can thus be considered a "presentation protein."

Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulator production of a suitable protein for structural studies.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure-function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation DeltaF508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N-1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.

Interest of colchicine for the treatment of cystic fibrosis patients. Preliminary report.

Cystic fibrosis (CF) lung disease is characterized by persistent inflammation. Antiinflammatory drugs, such as corticosteroids and ibuprofen, have proved to slow the decline of pulmonary function although their use is limited because of frequent adverse events. We hypothesized that colchicine could be an alternative treatment because of its antiinflammatory properties and upregulatory effect on cystic fibrosis transmembrane regulator (CFTR) closely related proteins. We herein present results obtained in an open study of eight CF children treated with colchicine for at least 6 months. Clinical status was better in all patients and respiratory function tests significantly improved in five. Median duration of antibiotherapy decreased significantly. These preliminary results support our hypothesis of a beneficial effect of colchicine in CF patients and stress the need for a controlled therapeutic trial.

NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein.

The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure. The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography. Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element. They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity. Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion. To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B. Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream. The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues. This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.

Insight into cystic fibrosis by structural modelling of CFTR first nucleotide binding fold (NBF1).

Cystic fibrosis is a human monogenic genetic disease caused by mutations in the cystic fibrosis (CF) gene, which encodes a membrane protein which functions as a channel: the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most frequent mutation, a deletion of phenylalanine F508 (delta F508), is located in the first nucleotide binding domain of CFTR: NBF1. This mutation leads to a folding defect in NBF1, responsible for an incomplete maturation of CFTR. The absence of CFTR at the surface of epithelial cells causes the disease. Determination of the three-dimensional (3D) structure of NBF1 is a key step to understanding the alterations induced by the mutation. In the absence of any experimental data, we have chosen to build a 3D model for NBF1. This model was built by homology modelling starting from F1-ATPase, the only protein of known 3D structure in the ATP binding cassette (ABC) family. This new model defines the central and critical position of F508, predicted in the hydrophobic core of NBF1. F508 indeed could be involved in hydrophobic interactions to ensure a correct folding pathway. Moreover, this model enables the localization of the LSGGQ sequence (a highly conserved sequence in the ABC family) in a loop, at the surface of the protein. This reinforces the hypothesis of its role for mediation of domain-domain interactions of functional significance for the channel regulation. Finally, the model also allows redefinition of the ends of NBF1 within the CFTR sequence. These extremities are defined by the secondary structure elements that are involved in the NBF1 fold. They lead to reconsideration of the C-terminal limit which was initially defined by the end of exon 12.

Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that the inter-domain region is disordered in solution.

Initiation factor IF3 from Escherichia coli plays a critical role in the selection of the correct initiation codon. This protein is composed of two domains, connected by a lysin-rich hydrophilic linker. The conformation of native IF3 was investigated by heteronuclear NMR spectroscopy. The two domains are independent and show little or no interaction. Heteronuclear relaxation studies of a sample selectively labelled on lysine residues demonstrates that the inter-domain linker is highly flexible, exhibiting increased 15N T2 values and negative 1H[15N] nuclear Overhause effects over a length of at least eight residues. Analysis of the rotational correlation times further shows that the motions of the two domains are most likely uncorrelated. The inter-domain linker thus displays almost totally unrestricted motions. Accordingly, the amide protons in the central region are shown to be in fast exchange with water. Such a high degree of flexibility of the inter-domain linker might be required for IF3 domains to interact with distant regions of the ribosome.

Three-dimensional solution structure of human angiogenin determined by 1H,15N-NMR spectroscopy--characterization of histidine protonation states and pKa values.

Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel formation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy. 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds. The backbone atomic rms difference from the mean coordinates is 0.067 +/- 0.012 nm and 0.13 nm from the previously determined crystal structure. The side-chain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues. The side-chain of His114 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than His114, with pKa values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pKa lower than 4.

What function for human lithostathine?: structural investigations by three-dimensional structure modeling and high-resolution NMR spectroscopy.

Human lithostathine is a 144-residue protein, expressed in various organs and pathologies. Several biological functions have been proposed for this protein. Among others, inhibition of nucleation and growth of CaCO3 crystals in the pancreas and bacterial aggregation has retained attention, because lithostathine presents high sequence similarities with calcium-dependent (or C-type) lectins. To study its structure-function relationship and compare it with that of C-type lectins, we have built a model for lithostathine. This model is derived from the only two C-type lectins of known structures: rat mannose binding protein and human E-selectin. An original strategy, inspired by that proposed by Havel and Snow, was designed for model building. We have undertaken NMR studies on the natural protein. Although complete structure determination has not yet been achieved, the NMR studies did confirm the main characteristics of the model. From analysis of the proposed model, we concluded that lithostathine is not expected to present sugar- or calcium-binding properties. Therefore, the mechanisms of bacterial aggregation and inhibition of CaCO3 nucleation and growth have not yet been elucidated.

Solution structure of bovine angiogenin by 1H nuclear magnetic resonance spectroscopy.

The three-dimensional structure of bovine angiogenin has been determined using two- and three-dimensional proton NMR spectroscopy. The solution structure is very close to that recently determined by X-ray diffraction analysis. This structure appears well defined, even if five loops and one helix exhibit greater flexibility. Analysis of the active site geometry confirms the position of the Glu-118 residue which obstructs the pyrimidine binding site. There is no experimental evidence of an unobstructed conformation of angiogenin in solution. In addition, it appears that the Glu-118 and Ser-119 residues and the cell receptor binding loop may play an important role in the differences of C-terminal fragment organization and ribonucleolytic activity observed between angiogenins and ribonuclease A.

1H, 15N resonance assignment and three-dimensional structure of CYP1 (HAP1) DNA-binding domain.

CYP1(HAP1) is a transcriptional activator involved in the aerobic metabolism of the yeast Saccharomyces cerevisiae. The amino acid sequence of its DNA-binding domain suggests that it belongs to the "zinc cluster" class. This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues. Structures of two such domains, those from GAL4 and PPR1, have been solved as complexes with DNA. These domains consist of the zinc cluster connected to a dimerization helix by a linker peptide. They recognize, as a dimer, an inverted repeat of a CGG motif that is separated by a specific number of bases. Interestingly, the specificity of that interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune between the structure of the linker peptide and the number of base-pairs separating the two CGGs. However, the CYP1 target sites fail to display such a consensus sequence. One of the two CGG sites is poorly conserved and some experiments suggest a direct rather than an inverted repeat. Using 1H, 15N and 113Cd NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment that consists of the zinc-cluster region, the linker peptide and a part of the dimerization helix. We have demonstrated that the six cysteine residues of the peptide chelate two cadmium ions as in GAL4 and PPR1. Fifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the linker peptide and the dimerization helix being unstructured under the conditions of our study. This region possesses the same overall fold as in GAL4 and PPR1, and most of the side-chains involved in the interaction with DNA are structurally conserved. This suggests that the CYP1 zinc-cluster region recognizes a CGG triplet in the same way as GAL4 and PPR1. In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix.

Amplification of radiation damping in a 600-MHz NMR spectrometer: Application to the study of water-protein interactions.

A new application of a recently developed electronic radiation-damping (RD) control system is presented. It is possible to amplify radiation damping so as to make the water magnetization return back to its equilibrium direction in a time shorter than the characteristic RD time. Certain types of experiments involving radiation damping as a selective inversion pulse can be significantly improved by this new method. Moreover, amplification of RD is shown to improve water suppression and consequently the dynamics of 2D NOESY experiments on proteins.

Solution structure of the ribosome-binding domain of E. coli translation initiation factor IF3. Homology with the U1A protein of the eukaryotic spliceosome.

Initiation of translation in prokaryotes requires the formation of a complex between the messenger RNA, the 30 S ribosomal subunit and the initiator tRNA(fMet). Initiation factor IF3 binds to the 30 S ribosomal subunit and proof-reads the initiation complex, thereby ensuring the accuracy of this step. IF3 also plays a pleiotropic role in the regulation of translation, as a result of differential influences exerted on the levels of the initiation of translation of genes or groups of genes. IF3 is composed of two independent domain or roughly identical sizes. We have expressed and purified the C-terminal domain of E. coli IF3 and shown that it retains both the 30 S particle binding and 70 S ribosome dissociating activities of the native protein. We have obtained 1H and 15N NMR resonance assignments and its 3D solution structure was calculated using 551 restraints. It is composed of a mixed beta-sheet backed by two alpha-helices. It shows a striking resemblance to the U1A small nuclear ribonucleoprotein structure, which binds to the U1 snRNA in the eukaryotic spliceosome. This suggests a convergent evolution process for these two proteins that are associated with ribonucleoproteic complexes.

1H and 15N resonance assignments and structure of the N-terminal domain of Escherichia coli initiation factor 3.

Initiation factor IF3 from Escherichia coli is composed of two domains connected by a hydrophilic peptide. In this study, the N-terminal domain (residues 7-83) has been overexpressed, 15N labelled and purified. NMR assignments for this domain have been obtained by two-dimensional and three-dimensional heteronuclear and homonuclear spectroscopy. Using distance geometry and simulated annealing, a three-dimensional solution structure was calculated using 506 NOE and 56 dihedral angle restraints. The resulting structure is composed of a five-stranded antiparallel beta sheet surrounded by two alpha helices. Since the heteronuclear 1H-15N correlation spectrum of the N-terminal domain of IF3 is an almost exact subset of that of the native protein, the assignments obtained and the structure calculated should be directly transposable to the full-length protein.

Neutralization of radiation damping by selective feedback on a 400 MHz NMR spectrometer.

Radiation damping is a phenomenon well known among NMR spectroscopists of proteins as a source of undesirable features, especially in high-field and high-Q probe NMR. In this paper, we present an electronic neutralization network which dramatically reduces radiation damping. It detects the radiation field profile and feeds back into the probe an rf field with identical amplitude and opposite phase. Experimental results of a practical implementation carried out on a 400 MHz Bruker spectrometer are shown.

The DNA-binding domain of the yeast Saccharomyces cerevisiae CYP1(HAP1) transcription factor possesses two zinc ions which are complexed in a zinc cluster.

Various fragments of the N-terminal, DNA-binding domain of the yeast Saccharomyces cerevisiae transcriptional activator CYP1(HAP1) have been cloned and expressed in Escherichia coli. The corresponding polypeptides have been analysed biochemically and we have undertaken a more extensive physical study of a fragment consisting of amino acids 49-126 [CYP1(49-126)]. We show that this CYP1(49-126) peptide requires zinc or cadmium in the growth medium in order to maintain a stable structure. A method to purify CYP1(49-126) is presented. We demonstrate that the purified CYP1(49-126) fragment contains two zinc ions/fragment or two cadmium ions/fragment, which are necessary for DNA binding. 113Cd one-dimensional NMR data suggest that CYP1(HAP1) has a tetrahedral coordination, and that it forms a zinc-cluster complex like GAL4.

Proton resonance assignments and secondary structure of bovine angiogenin.

Angiogenins are 14-kDa proteins able to induce blood vessel growth in various preparations and are thus thought to be involved in the development of solid tumors. Angiogenins have significant similarities with extracellular ribonuclease and possess a characteristic nuclease activity against large RNA molecules. These proteins are also able to induce second-messenger pathways. We have undertaken the determination of the three-dimensional structure of bovine angiogenin by using nuclear magnetic resonance (NMR) spectroscopy. Since this protein was directly purified from cow milk, it was not possible to enrich angiogenin with 13C or 15N isotopes. However, extensive use of two-dimensional and three-dimensional proton NMR experiments enabled us to identify all but four spin systems and to assign all corresponding proton resonances. Identification of most backbone-backbone nuclear Overhauser enhancements led to the characterization of the secondary structure elements of the protein. Comparison with the structure of ribonuclease A and analysis of the location of conserved residues confirmed that the two molecules have very similar structures.

Bayesian signal extraction from noisy FT NMR spectra.

The statistical interpretation of the histogram representation of NMR spectra is described, leading to an estimation of the probability density function of the noise. The white-noise and Gaussian hypotheses are discussed, and a new estimator of the noise standard deviation is derived from the histogram strategy. The Bayesian approach to NMR signal detection is presented. This approach homogeneously combines prior knowledge, obtained from the histogram strategy, together with the posterior information resulting from the test of presence of a set of reference shapes in the neighbourhood of each data point. This scheme leads to a new strategy in the local detection of NMR signals in 2D and 3D spectra, which is illustrated by a complete peak-picking algorithm.

Alpha-cobratoxin: proton NMR assignments and solution structure.

The solution structure of alpha-cobratoxin, a neurotoxin purified from the venom of the snake Naja naja siamensis, at pH 3.2 is reported. Sequence-specific assignments of the NMR resonances was attained by a combination of a generalized main-chain-directed strategy and of the sequential method. The NMR data show the presence of a triple-stranded beta-sheet (residues 19-25, 36-41, and 52-57), a short helix, and turns. An extensive number of NOE cross peaks were identified in the NOESY NMR maps. These were applied as distance constraints in a molecular modeling protocol which includes distance geometry and dynamical simulated annealing calculations. A single family of structures is observed which fold in such a way that three major loops emerge from a globular head. The solution and crystal structures of alpha-cobratoxin are very similar. This is in clear contrast to results reported for alpha-bungarotoxin where significant differences exist.