Nuclear magnetic resonance analysis of protein-DNA interactions.

Recent methodological and instrumental advances in solution-state nuclear magnetic resonance have opened up the way to investigating challenging problems in structural biology such as large macromolecular complexes. This review focuses on the experimental strategies currently employed to solve structures of protein-DNA complexes and to analyse their dynamics. It highlights how these approaches can help in understanding detailed molecular mechanisms of target recognition.

Assessment of the performance of the Ames II assay: a collaborative study with 19 coded compounds.

Nineteen coded chemicals were tested in an international collaborative study for their mutagenic activity. The assay system employed was the Ames II Mutagenicity Assay, using the tester strains TA98 and TAMix (TA7001-7006). The test compounds were selected from a published study with a large data set from the standard Ames plate-incorporation test. The following test compounds including matched pairs were investigated: cyclophoshamide, 2-naphthylamine, benzo(a)pyrene, pyrene, 2-acetylaminofluorene, 4,4'-methylene-bis(2-chloroaniline), 9,10-dimethylanthracene, anthracene, 4-nitroquinoline-N-oxide, diphenylnitrosamine, urethane, isopropyl-N(3-chlorophenyl)carbamate, benzidine, 3,3'-5,5'-tetramethylbenzidine, azoxybenzene, 3-aminotriazole, diethylstilbestrol, sucrose and methionine. The results of both assay systems were compared, and the inter-laboratory consistency of the Ames II test was assessed. Of the eight mutagens selected, six were correctly identified with the Ames II assay by all laboratories, one compound was judged positive by five of six investigators and one by four of six laboratories. All seven non-mutagenic samples were consistently negative in the Ames II assay. Of the four chemicals that gave inconsistent results in the traditional Ames test, three were uniformly classified as either positive or negative in the present study, whereas one compound gave equivocal results. A comparison of the test outcome of the different investigators resulted in an inter-laboratory consistency of 89.5%. Owing to the high concordance between the two test systems, and the low inter-laboratory variability in the Ames II assay results, the Ames II is an effective screening alternative to the standard Ames test, requiring less test material and labor.

Solution structure of the N-terminal domain of the human TFIIH MAT1 subunit: new insights into the RING finger family.

The human MAT1 protein belongs to the cyclin-dependent kinase-activating kinase complex, which is functionally associated to the transcription/DNA repair factor TFIIH. The N-terminal region of MAT1 consists of a C3HC4 RING finger, which contributes to optimal TFIIH transcriptional activities. We report here the solution structure of the human MAT1 RING finger domain (Met(1)-Asp(65)) as determined by (1)H NMR spectroscopy. The MAT1 RING finger domain presents the expected betaalphabetabeta topology with two interleaved zinc-binding sites conserved among the RING family. However, the presence of an additional helical segment in the N-terminal part of the domain and a conserved hydrophobic central beta strand are the defining features of this new structure and more generally of the MAT1 RING finger subfamily. Comparison of electrostatic surfaces of RING finger structures shows that the RING finger domain of MAT1 presents a remarkable positively charged surface. The functional implications of these MAT1 RING finger features are discussed.

Structural analysis of WW domains and design of a WW prototype.

Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.

Solution structure of N-(2-deoxy-D-erythro-pentofuranosyl)urea frameshifts, one intrahelical and the other extrahelical, by nuclear magnetic resonance and molecular dynamics.

The presence of a N-(2-deoxy-D-erythro pentofuranosyl)urea (henceforth referred to as deoxyribosylurea) residue, ring fragmentation product of a thymine, in a frameshift situation in the sequence 5'd(AGGACCACG).d(CGTGGurTCCT) has been studied by 1H and 31P nuclear magnetic resonance and molecular dynamics. At equilibrium, two species are found in slow exchange. We observe that the deoxyribosylurea residue can be either intra- or extrahelical within structures which otherwise do not deviate strongly from that of a B-DNA as observed by NMR. Our study suggests that this is determined by the nature and number of hydrogen bonds which this residue can form as a function of two possible isomers. There are two possible structures for the urea side chain, either cis or trans for the urido bond which significantly changes the hydrogen bonding geometry of the residue. In the intrahelical species, the cis isomer can form two good hydrogen bonds with the bases on the opposite strand in the intrahelical species, A4 and C5, which is not the case for the trans isomer. This results in a kink in the helical axis. For the major extrahelical species, the situation is reversed. The trans isomer is able to form two good hydrogen bonds, with G13 on the same strand and A7 on the opposite strand. For the extrahelical species, the cis isomer can form only one hydrogen bond. In this major structure the NMR data show that the bases which are on either side of the deoxyribosylurea residue in the sequence, G14 and T16, are stacked over each other in a way similar to a normal B-DNA structure. This requires the formation of a loop for the backbone between these two residues. This loop can belong to one of two families, right- or left-handed. In a previous study of an abasic frameshift [Cuniasse et al. (1989) Biochemistry 28, 2018-2026], a left-handed loop was observed, whereas in this study a right-handed loop is found for the first time in solution. The deoxyribosylurea residue lies in the minor groove and can form both an intra- and an interstrand hydrogen bond.

NMR investigations of the role of the sugar moiety in glycosylated recombinant human granulocyte-colony-stimulating factor.

Human granulocyte-colony-stimulating factor (G-CSF) is a hematopoietic growth factor that plays a major role in the stimulation of the proliferation and maturation of granulocyte neutrophil cells. With the recent increased understanding of its biological properties in vivo together with available preparations of recombinant human G-CSF, this growth factor has become an essential agent for clinical applications. The presence of an O-linked carbohydrate chain at position 133 greatly improves the physical stability of the protein. To clarify the molecular basis for the stabilisation effect of saccharide moieties on human G-CSF the whole glycoprotein expressed in CHO cells has been investigated by means of two 1H-NMR-spectroscopy and two 1H-detected-heteronuclear 1H-13C experiments at natural abundance, and compared with the non-glycosylated form. The present NMR study reports assignments of 1H and 13C resonances of the bound saccharidic chain NeuNAc(alpha2-3)Gal(beta1-3)[NeuNAc(alpha2-6)]GalNAc, where NeuNAc represents N-acetylneuraminic acid, and demonstrates the alpha-anomeric configuration of the N-acetylgalactosamine-threonine linkage. It also provides results suggesting that the carbohydrate moiety reduces the local mobility around the glycosylation site, which could be responsible for the stabilising effect observed on the glycoprotein.

Solution structure of two mismatches A.A and T.T in the K-ras gene context by nuclear magnetic resonance and molecular dynamics.

Two mismatches, one homopurine (A.A) and the other homopyrimidine (T.T), have been incorporated at the central position N of: 5'd(GCCACNAGCTC).d(GAGCTNGTGGC) in order to study nuclear magnetic resonance spectra and molecular dynamics. These duplexes constitute the sequence 29-39 of the K-ras gene coding for Gly12, a hot spot for mutation. The NMR spectra show that the duplexes are not greatly distorted by the introduction of the mismatches and their global conformation is that of a canonical B-form double helix. For both systems, no structural change is observed in the pH range 4.7-9. For the duplex containing the homopurine A.A mismatch, we propose a type of pairing involving one hydrogen bond between the amino group of one central adenine and the nitrogen N1 of the opposite adenine. For the duplex containing the mispaired T.T bases, NMR spectra recorded in H2O at 282 K indicate that these central bases are engaged in wobble pairing, involving two imino-carbonyl hydrogen bonds. For both systems two conformations with the same donor and acceptor pattern can coexist, one being obtained from the other by a 180 degrees rotation about the pseudodyadic axis. Exchange between the two forms is observed by NMR at low temperature for the T.T mispair and also inferred from NMR measurements on the A.A system. The presence of this exchange and its pathway has been investigated by molecular dynamics calculations on both systems. Distance restrained and unrestrained molecular dynamics are in very good agreement with the NMR data. The average structure for either mispair shows only small conformational change from normal B DNA. For each, a systematic pathway is observed for exchange between the two conformations.

Solution conformation of an oligonucleotide containing a urea deoxyribose residue in front of a thymine.

Urea residues are produced by ionizing radiation on thymine residues in DNA. We have studied an oligodeoxynucleotide containing a thymine opposite the urea residue, by one and two dimensional NMR spectroscopy. The urea deoxyribose exists as two isomers with respect to the orientation about the peptide bond. For the trans isomer we find that the thymine and urea site are positioned within the helix and are probably hydrogen bonded. The oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. A minor species is observed, in which the urea deoxyribose and the opposite base adopt an extrahelical position and this corresponds to the isomer cis for the peptide bond.