HIV-1 integrase and virus and cell DNAs: complex formation and perturbation by inhibitors of integration.

HIV-1 integrase (IN) catalyzes integration of viral DNA into cell DNA through 3'-processing of viral DNA and strand transfer reactions. To learn on binding of IN to DNAs and IN inhibition we applied spectroscopy (circular dichroism, fluorescence) in a simplified model consisting in a peptide analogue (K156) of alpha4 helix involved in recognition of viral and cell DNA; an oligonucleotide corresponding to the U5' LTR DNA end; and an inhibitor (TB11) of the diketo acid (DKA) family. Results extrapolated to IN show that: the enzyme binds viral DNA with high affinity and specificity, but cell DNA with low affinity and specificity; the affinity of TB11 for IN is high enough to impair the binding of IN to cell DNA, but not to viral DNA. This explains why TB11 is an inhibitor of strand transfer but not of 3'-processing. These results can help in the search of new IN inhibitors.

Peptide inhibitors of HIV-1 integrase dissociate the enzyme oligomers.

Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.

Impact of CpG methylation on structure, dynamics and solvation of cAMP DNA responsive element.

Methylation of CpG motifs in DNA is involved in the control of gene expression and in several other epigenic effects. It suppresses also the immuno-stimulation properties of bacterial or viral DNAs that contain CPGS: However, effects of methylation on the DNA structure and dynamics are not clear. Here we carried out a 10 ns MD simulation, confronted to an NMR analysis, of a hexadecanucleotide with the cAMP responsive element (CRE) DNA methylated at its center: d(GAGATGAmCGTCATCTC)(2) (CREmet). Methylation does not introduce significant structure modification but reduces the dynamics. Molecular mechanics and generalized Born solvation energy calculations showed that the stiffness of CREmet arises from both a restriction of the conformational space by the bulky methyl groups and a folding of DNA around the hydrophobic methyls. The latter effect is favored when the GpA steps belonging to the TGA binding half-sites adopt the BII conformation. The inability of the methylated DNAs to interact with their protein partners-either transcription factors for gene regulation or a Toll-like receptor for immunostimulation-could result from both the obstacle created by methyls, preventing crucial interactions, and the loss of DNA flexibility, reducing its adaptability. Results are discussed in the light of NMR and crystallographic data.

Self-association of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by electro spray ionization mass spectrometry in trifluoroethanol/water mixtures.

Establishing the auto-associative properties of a molecule in solution can be important for determination of its structure and function. EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) has been designed to inhibit HIV-1 integrase via formation of a stable coiled-coil structure with a nearly homologous segment in the enzyme. The latter catalyzes the permanent incorporation of a DNA copy of the retrovirus genome into host cell DNA, and is thus essential to the life of the retrovirus. This makes integrase an obvious drug target in the therapy of AIDS. The present work has demonstrated, using electrospray ionization mass spectrometry (ESI-MS), that EAA26 is monomeric in pure water, and tetrameric and dimeric at respectively low and medium concentrations of 2,2,2-trifluoroethanol (TFE), and again monomeric at higher TFE concentrations. Thus, the apolar solvent TFE may contribute to either stabilization or disruption of the intermolecular hydrophobic contacts depending on its concentration in aqueous solution. Previous NMR and ultracentifugation results are thus confirmed, indicating the reliability of ESI-MS for defining the self-association state of biologically relevant peptides in both water and organic-water solutions.

NMR study of a heterochiral DNA hairpin:impact of L-enantiomery in the loop.

We carried out a structural study of the DNA heterochiral strand d (AGCTTATCAT(L)CGATAAGCT), -AT(L)C-, where T(L) (L thymine ) replaces T (natural D-thymine). -AT(L)C- is a structural analog of -ATC- that belongs to a strong topoisomerase II DNA cleavage site and which has been shown to resolve into a hairpin structure with a stem formed by eight Waston-Crick base-pairs and a single residue loop closed by an A.C sheared base-pair. Although - AT(L)C-, like its parent -ATC-, folds into a hairpin structure at low and high DNA concentrations it displays a lower stability (Tm of 56 degrees C versus 58.5 degrees C). Several NMR features in -AT(L)C- account for the disruption of the A.C pairing in the loop and a weakening of the C.G base-pair stability at the stem-loop junction. For instance, the exchange of the loop imino protons with solvent is accelerated compared with the natural oligonucleotide -ATC-. The higher flexibility of the heterochiral loop is confirmed by the results of NMR restrained molecular dynamics. In the calculated final structures of -AT(L)C-, the T10(L) residue moves the A9 and C11 residues away, thus preventing the loop closure through a C.A sheared base-pair and the achievement of a good base-base or sugar-base stacking. Actually, most of the stabilizing interactions present in -ATC- are lost in the heterochiral - AT(L)C- explaining its weaker stability.

A two B-Z junction containing DNA resolves into an all right-handed double-helix.

Natural and artificial oligonucleotides are capable of assuming many different conformations and functions. Here we present results of an NMR restrained molecular modelling study on the conformational preferences of the modified decanucleotide d((m)C1G2(m)C3G4C5(L)G6(L)(m)C7G8(m)C9G10) .d((m)C11G12(m)C13G14C15(L)G (L)16(m)C17-G18(m)C19G20 ) which contains L deoxynucleotides in its centre. This chimeric DNA was expected to form a right-left-right-handed B-type double-helix (BB*B) at low salt concentration. Actually, it matured into a fully right-handed double helix with its central C(L)pG(L) core forming a right-handed Z-DNA helix embedded in a B-DNA matrix (BZ*B). The interplay between base-base and base-sugar stackings within the core and its immediately adjacent residues was found to be critical in ensuring the stabilisation of the right-handed helix. The structure could serve as a model for the design of antisense oligonucleotides resistant to nucleases and capable of hybridising to natural DNAs and RNAs.

Bending and adaptability to proteins of the cAMP DNA-responsive element: molecular dynamics contrasted with NMR.

DNA bending is assumed to play a crucial role during recognition of the cAMP-responsive element (CRE) by transcription factors. However, diverging results have been obtained for the bending direction of the unbound double helix. The refined NMR structures present a bend directed toward the minor groove, while biochemical methods conclude that there is a bend toward the major groove. The present 10-ns molecular dynamics (MD) simulation of d(GAGATGACGTCATCTC)(2), which contains the octamer CRE in its center, was carried out with AMBER in explicit water and counterions. It shows that CRE is a flexible segment, although it is bent slightly toward the major groove (7 degrees -8 degrees ) on the average. The MD structure agrees with both the biochemical results and unrefined NMR data. The divergence with the NMR refined structures suggests an improper electrostatic parameterization in the refinement software. The malleability of the central CpG is certainly the major contribution to the curving of the whole CRE segment in both the unbound and bound states. Comparison with the crystal structure of CRE bound to GCN4 shows that the deformation induced by the protein is concentrated mainly on the CpG step, rendering the bound structure of CRE closer to the structure of the 12-0 tetradecanoylphorbol-beta-acetate-responsive element.

A DNA hairpin with a single residue loop closed by a strongly distorted Watson-Crick G x C base-pair.

Our previous NMR and modeling studies have shown that the single-stranded 19mer oligonucleotides d(AGCTTATC-ATC-GATAA GCT) -ATC- and d(AGCTTATC-GAT-GATAAGCT) -GAT- encompassing the strongest topoisomerase II cleavage site in pBR322 DNA could form stable hairpin structures. A new sheared base-pair, the pyrimidine-purine C x A, was found to close the single base -ATC- loop, while -GAT- displayed a flexible loop of three/five residues with no stabilizing interactions. Now we report a structural study on -GAC-, an analog of -GAT-, derived through the substitution of the loop residue T by C. The results obtained from NMR, non-denaturing PAGE, UV-melting, circular dichroism experiments and restrained molecular dynamics indicate that -GAC- adopts a hairpin structure folded through a single residue loop. In the -GAC- hairpin the direction of the G9 sugar is reversed relative to the C8 sugar, thus pushing the backbone of the loop into the major groove. The G9 x C11 base-pair closing the loop is thus neither a sheared base-pair nor a regular Watson-Crick one. Although G9 and C11 are paired through hydrogen bonds of Watson-Crick type, the base-pair is not planar but rather adopts a wedge-shaped geometry with the two bases stacked on top of each other in the minor groove. The distortion decreases the sugar C1'-C1' distance between the paired G9 and C11, to 8 A versus 11 A in the standard B-DNA. The A10 residue at the center of the loop interacts with the G9 x C11 base-pair, and seems to contribute to the extra thermal stability displayed by -GAC- compared to -GAT-. Test calculations allowed us to identify the experimental NOEs critical for inducing the distorted G.C Watson-Crick base-pair. The preference of -GAC- for a hairpin structure rather than a duplex is confirmed by the diffusion constant values obtained from pulse-field gradient NMR experiments. All together, the results illustrate the high degree of plasticity of single-stranded DNAs which can accommodate a variety of turn-loops to fold up on themselves.

Self-association and domains of interactions of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by analytical ultracentrifugation and NMR experiments in trifluoroethanol/H(2)O mixtures.

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.

An NMR and molecular modelling analysis of d(CTACTGCTTTAG). d(CTAAAGCAGTAG) reveals that the particular behaviour of TpA steps is related to edge-to-edge contacts of their base-pairs in the major groove.

In a previous NMR study we detected the presence of particular motions and hydration properties within the DNA fragment d(CTACTGCTTTAG).d(CTAAAGCAGTAG). Now, we report on an NMR and molecular modelling analysis of this sequence focusing our attention on the biologically important TpA steps. NOe and coupling constant restraints were introduced in three different modelling protocols: X-PLOR and JUMNA used with Flex and AMBER94 as force-fields. Despite their differences the protocols produce similar mean B-DNA structures (r.m.s.d. <1 A). The new information confirms our previous experimental results on the narrowing of the minor groove along the T8T9T10/A17A16A15 run and the sudden widening at the T10pA11 step ending this run. It is further shown that this step displays a large positive roll with its T10:A15 and A11:T14 base-pairs likely stabilised by amino-amino and amino-carbonyl interactions in the major groove. A relationship between roll values and amino-amino and amino-carbonyl distances strongly suggests that electrostatics or bifurcated hydrogen-bonds could be responsible for induction of positive rolls in TpA steps. Such edge-to-edge interactions could explain the slower motions shown by the adenine A15. The influence of these interactions on the stabilisation of particular DNA conformers is discussed using our data and those provided by the recent literature.

Sequence effects on energetic and structural properties of phosphorothioate DNA: a molecular modelling study.

Phosphorothioate (PS) oligonucleotides constitute a new class of potent drugs, resulting from the replacement of one anionic oxygen of the phosphodiester backbone by one sulphur atom. This replacement confers chirality to the phosphorus atom (PSS or PSR) and alters the energetic, structural and biological properties of B-DNA. These properties were assessed by molecular mechanics calculations on a set of regular sequences, d(YR)8.d(YR)8 and d(RR)8.d(YY)8 (R, purine; Y, pyrimidine). Results indicated: (i) destabilisation of both the PS(R)and the PSS oligomers, the loss of total energy being mainly due to a variation in the electrostatic term; (ii) an additional chirality effect, due to van der Waals and backbone angle energies, larger for PSS oligomers than for PSR oligomers; (iii) a clear sequence effect on stability, particularly from the base immediately preceding the PS group. Even though the PS group alters the stability of oligomers, it does not significantly modify the conformation. Altogether, our molecular modelling data parallel the available experimental data. Our results reveal that sequence effects on the energetic properties of PS oligomers are local and additive. Therefore, studies of the set of the 10 unique double-stranded modified dinucleotide steps included in regular oligomers could be used to predict the behaviour of any double-stranded PS-DNA.

Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

Conformational aspects of HIV-1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide.

Monospecific antibodies were raised against a synthetic peptide K159 (SQGVVESMNKELKKIIGQVRDQAEHLKTA) reproducing the segment 147-175 of HIV-1 integrase (IN). Synthesis of substituted and truncated analogs of K159 led us to identify the functional epitope reacting with antibodies within the C-terminal portion 163-175 of K159. Conformational studies combining secondary structure predictions, CD and NMR spectroscopy together with ELISA assays, showed that the greater is the propensity of the epitope for helix formation the higher is the recognition by anti-K159. Both the antibodies and the antigenic peptide K159 exhibited inhibitory activities against IN. In contrast, neither P159, a Pro-containing analog of K159 that presents a kink around proline but with intact epitope conformation, nor the truncated analogs encompassing the epitope, were inhibitors of IN. While the activity of antibodies is restricted to recognition of the sole epitope portion, that of the antigenic K159 likely requires interactions of the peptide with the whole 147-175 segment in the protein [Sourgen F., Maroun, R.G., Frère, V., Bouziane, A., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765-773]. Actually, of all tested peptides only K159 was found to fulfill condition of minimal number of helical heptads to achieve the formation of a stable coiled-coil structure with the IN 147-175 segment. The binding of antibodies and of the antigenic peptide to this segment of IN hampers the binding of IN to its DNA substrates in filter-binding assays. This appears to be the main effect leading to inhibition of integration. Quantitative analysis of filter-binding assay curves indicates that two antibody molecules react with IN implying that the enzyme is dimeric within these experimental conditions. Together, present data provide an insight into the structure-function relationship for the 147-175 peptide domain of the enzyme. They also strongly suggest that the functional enzyme is dimeric. Results could help to assess models for binding of peptide fragments to IN and to develop stronger inhibitors. Moreover, K159 antibodies when expressed in vivo might exhibit useful inhibitory properties.

An intrinsic curvature towards the minor groove in the cAMP-responsive element DNA found by combined NMR and molecular modelling studies.

The cAMP-responsive element (CRE, 5'-TGACGTCA-3') is essential to the transcriptional function of numerous gene promoters in eukaryotic cells. We carried out NMR restrained molecular mechanics studies using two different force fields (Flex and "AMBER94") on a hexadecanucleotide d(GAGATGACGTCATCTC) containing CRE. Results indicated that free CRE is a B-DNA that is intrinsically curved towards the minor groove. To our knowledge, NMR restraints have not previously been useful in accounting for a global DNA curvature. In order to validate the bend in CRE, we applied a new strategy in which DNA structures displaying different curvatures were generated and then compared with NMR data. Conformations of CRE curved towards the minor groove provided the best agreement with NMR data. Our results contrast with previous results obtained from NMR restrained modelling and gel methods; these suggested conformations that were straight or curved towards the major groove, respectively. The curve in free CRE is spread along the DNA helix: several kinks are repeated in phase within the helical turn, although they are centred mainly on CpG in between the TGA half-sites, thus slightly increasing their spacing within the major groove. Comparison with the crystal structure of CRE complexed to general control protein 4 showed that the curve orientation is reversed from the minor to the major groove upon protein binding, due to a helix distortion concentrated mainly on CpG.

Low-molecular-weight dextran derivatives (f-CMDB) enter the nucleus and are better cell-growth inhibitors compared with parent CMDB polymers.

Carboxymethyldextrans-benzylamide (CMDB) are dextran derivatives that are statistically substituted with carboxymethyl and benzylamide groups. These molecules display a variety of biological effects, one of which is their inhibitory activity against mammary tumor cell growth, both in vitro and in vivo. We and others have previously shown that the effects of CMDB on cell growth are related to their ability to interact with the growth factor FGF-2. The binding modifies the conformation of FGF-2, leading to the suppression of its mitogenic activity. Here, the method previously reported to fragment natural polysaccharide fucans has been applied to CMDB (80,000 g/mol). f-CMDB (fragmented CMDB) of molecular weights from 6000 to 20,000 g/mol were found to be more potent inhibitors of MCF7 mammary tumor cell growth than high-molecular-weight CMDB. Confocal microscopy experiments using CMDB and f-CMDB labeled with the fluorophore DTAF (5-([4,6-dichlorotriazine-2-yl]amino) fluorescein) indicate that only low-molecular-weight f-CMDB penetrate into the nucleus of MCF7 cells. It is thus assumed that the better inhibitory properties demonstrated by f-CMDB, compared with CMDB, are related to their better ability to penetrate the nucleus and interact with nuclear targets, including topoisomerase II. The DNA relaxation properties of the latter are inhibited in vitro by both CMDB and f-CMDB. These findings could help us to develop models of low-molecular-weight oligosaccharide derivatives exhibiting better antiproliferative and antitumor properties.

Comparative structural analysis by [1H,31P]-NMR and restrained molecular dynamics of two DNA hairpins from a strong DNA topoisomerase II cleavage site.

The structural analysis of two single-stranded DNAs d(AGCTTATCATCGATAAGCT) (ATC-19) and d(AGCTTATCGATGATAAGCT) (GAT-19) was performed by NMR and restrained molecular dynamics. These oligonucleotides reproduce the 15-33 segment of phage pBR322 DNA, which contains a strong cleavage site for topoisomerase II coupled to the antitumor drugs VP-16 and ellipticine. Because of their partial palindromic nature, the two oligonucleotides ATC-19 and GAT-19 may fold back into stable hairpin structures, consisting of a stem of eight base-pairs and a loop of three residues. NMR assignments and conformational parameters were determined from combined 2D NOESY, COSY and 1H-31P spectra. Conformations of ATC-19 and GAT-19 hairpins were calculated using the X-PLOR 3.1 program. Structures were generated through simulated annealing procedures starting from 50 structures with randomized torsion angles. A good convergence was observed for ATC-19 molecules, while no consensus was found for GAT-19. Within the GAT-19 loop, the base stacking was poor and no hydrogen bond could be detected. In contrast, ATC-19 displayed a well-defined three residue loop stabilized by both extensive base stackings and hydrogen bonding between the N3 atom of the adenine ring and the amino group of the cytosine ring. The results confirm our earlier ATC-19 structure obtained by a completely different calculation procedure (JUMNA) and the higher thermal stability of ATC-19 compared to GAT-19. Moreover, due to its mismatched base-pair, the ATC-19 loop may be better described as a single residue loop rather than a three residue loop. Comparison of this loop to those containing sheared purine.purine base-pairs revealed striking resemblances, particularly on the backbone angle combination. Finally, the differences observed between the ATC-19 and GAT-19 structures could help toward understanding the sequential cleavage of DNA strands by topoisomerase II.

Helical and coiled-coil-forming properties of peptides derived from and inhibiting human immunodeficiency virus type 1 integrase assessed by 1H-NMR--use of NH temperature coefficients to probe coiled-coil structures.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) which catalyzes viral DNA integration into the host genome of infected cells represents an attractive target for AIDS therapy. We have previously demonstrated the ability of the IN-(147-175)-peptide derived from the catalytic core domain of HIV-1 IN to inhibit the enzyme activity in vitro. IN-(147-175)-peptide contains four heptad repeats and displays a high propensity for coiled-coil formation while its [P159]IN-(147-175)-peptide analog (Lys159-->Pro in the protein, Lys13-->Pro in the peptide) is unable to form a stable coiled-coil and is devoid of inhibitory activity [Sourgen, F., Maroun, R. G., Frère, V., Bouziane, M., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765-773]. Now, we report results from an NMR study on IN-(147-175)-peptide and [P159]IN-(147- 175)-peptide as well as on an optimized [E156, A163, A167]IN-(147-175)-peptide that is a better inhibitor of IN than IN-(147-175)-peptide. While in aqueous solution, IN-(147-175)-peptide and [P159]IN-(147-175)-peptide display only nascent helical features, [E156, A163, A167]IN-(147-175)-peptide exhibits 20% of helical content. In 20% trifluoroethanol/80% H2O, the helix content is the highest for [E156, A163, A167]IN-(147-175)-peptide (approximately 70%) and the lowest for [P159]IN-(147-175)-peptide (approximately 40%), due to a local helix break caused by the Pro residue. The NHs of residues in the two central helical heptads (a-g) of IN-(147-175)-peptide and [E156, A163, A167]IN-(147-175)-peptide display a regular periodic variation of their temperature coefficients in 20% trifluoroethanol. The b, c and f residues on the hydrophilic face of the amphipathic helix show high coefficients reflecting hydrogen bonded NHs, while the a and d residues on the hydrophobic face exhibit low coefficients, near random-coil values. The particular arrangement of the hydrophobic side-chains of a and d residues at the coiled-coil interface reduces the access of trifluoroethanol molecules to their amide groups. The inability of trifluoroethanol molecules to create interactions with the amide C=O groups, these being required to strengthen the intrahelical C=O...H-N hydrogen bonds, is the main cause for observation of heptadic a and d residues with low NH temperature coefficients. Such effects concern mostly the two central helical heptads of IN-(147-175)-peptide and [E156, A163, A167]IN-(147-175)-peptide implying that these ones are engaged in stable parallel coiled coils. Our results provide a link between the propensity of peptides for helix formation, their coiled-coil properties and their efficiency to inhibit IN.

Flexibility of the B-DNA backbone: effects of local and neighbouring sequences on pyrimidine-purine steps.

The structurally correlated dihedral angles epsilon and zeta are known for their large variability within the B-DNA backbone. We have used molecular modelling to study both energetic and mechanical features of these variations which can produce BI/BII transitions. Calculations were carried out on DNA oligomers containing either YpR or RpY dinucleotides steps within various sequence environments. The results indicate that CpA and CpG steps favour the BI/BII transition more than TpA or any RpY step. The stacking energy and its intra- and inter-strand components explain these effects. Analysis of neighbouring base pairs reveals that BI/BII transitions of CpG and CpA are easiest within (Y)n(R)n sequences. These can also induce a large vibrational amplitude for TpA steps within the BI conformation.

An NMR study of d(CTACTGCTTTAG).d(CTAAAGCAGTAG) showing hydration water molecules in the minor groove of a TpA step.

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-H1'(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment (approximately 3.9A) and a sudden widening at the T10:A15 base-pair (approximately 5.3A), the standard B-DNA distance being approximately 5A. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n> or =2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for A11 ending the 5'TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the A11:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to A11 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.

Peptide fragments of DNA topoisomerase II with helix-forming and coiled-coil-forming properties act as inhibitors of the enzyme.

We have previously shown that a synthetic peptide (dL) consisting of amino acids 1013-1056 of human alpha topoisomerase II adopted an alpha-helix structure and formed a stable dimer coiled-coil in solution [Frère, V., Sourgen. F., Monnot, M., Troalen, F. & Fermandjian, S. (1995) J. Biol. Chem. 270, 17502-17507]. Here we studied two peptides, dP and dLshort, which are related to dL but which have a double substitution Leu1026-->Pro, Leu1037-->Pro and a deletion of the 15 C-terminal residues, respectively. The peptides were studied for their ability to form alpha-helix structures, coiled coils, and to inhibit topoisomerase II activity. In combining circular dichroism spectra with AGADIR prediction for helix structures, we demonstrated that the dLshort peptide, like its parent dL peptide, adopts an alpha-helix structure and can autoassociate into coiled-coils, while dP is completely devoid of such properties. Remarkably, only the dL and dLshort peptides act as good inhibitors of topoisomerase II in various in vitro assays. However, the dLshort peptide has a stronger helix potential and behaves as a much more potent inhibitor (5 microM versus 200 microM) compared to the dL peptide. All these data strongly suggest that the greater inhibitory effect demonstrated by the dLshort peptide is related to its higher ability to form a stable amphiphilic helix, which in turn better recognizes its homologous helical segment in topoisomerase II. Finally, we propose that the dL and the dLshort peptides could interfere with the enzymatic activity of topoisomersase II in modifying its autoassociation or translocation properties. Such peptides may serve as useful models for developing simpler and more specific inhibitors of topoisomerase II.