Molecular modeling of a Leishmania antigen eIF-4A: identification of a potential epitope implicated in the adjuvant effect.

In leishmaniasis, the development of an effective vaccine depends on its capacity to elicit Th1 immune responses. Despite many approaches, recent vaccines do not induce sufficient levels of protection and long-term memory. To overcome this problem, vaccines are used with adjuvants that drive immunity towards Th1 and enhance endogenous production of IL-12, a Th1-promoting cytokine. Although exogenous IL-12 may act as an effective adjuvant, it has an elevated cost and toxic effects. Therefore, it is important to develop cheap and safer adjuvants that trigger endogenous IL-12. Of particular interest is LmeIF a unique Leishmania protein that provides significant adjuvant effects by stimulating high IL-12 production. This investigation was designed to identify the structural factors responsible for the adjuvant effects of LmeIF by establishing the 3D models of LmeIF and MueIF (mouse) by homology modeling based on the X-ray structure of their homologs in yeast and comparing their stereo-electronic properties. The molecular electrostatic potential was computed around each model and used to localize the active site and the most different amino acids between LmeIF and MueIF. Sequence alignment of LmeIF with eIF-4 from other species showed three residues (Q186, A189, E214) in the active site which were peculiar to the Leishmania protein. Long MD simulation was carried out on LmeIF fragment 129-261 to compare its folding with the native protein. Despite a high degree of sequence similarity with different species, we have identified in LmeIF a set of residues unique to the protozoan parasite Leishmania which may be potentially responsible for its adjuvant property. Using LmeIF model, a plausible surface region for binding with its receptor was also identified.

Plausible interaction of an alpha-fetoprotein cyclopeptide with the G-protein-coupled receptor model GPR30: docking study by molecular dynamics simulated annealing.

In this manuscript, the procedure of molecular dynamics simulated annealing is applied to locate a probable receptor and binding site of a cyclicpeptide that inhibits estrogen-stimulated proliferation of breast cancer. The hydrophilic cyclopeptide EMTOVNOGQ (O = 4-hydroxyproline), derived from alpha-fetoprotein, is an inhibitor of estrogen-stimulated proliferation of human breast cancer. This peptide has been shown to act through a mechanism different from that of estrogen; however, its receptor is unknown. We report computer experiments that suggest that this peptide may execute its actions by interacting with GPR30, a G-protein-coupled receptor. The subject of this work is the simulation, by molecular dynamics simulated annealing, of the interaction of cyclopeptide EMTOVNOGQ with receptor GPR30 protein. A conformational analysis of the cyclopeptide was undertaken and the final structure was docked on several sites of the GPR30 3D model. Our results show that the cyclopeptide interacts on the pocket located between TM6 and TM7 transmembrane helices of the G-protein, triggering a slight conformational change in the secondary structure of the receptor in the complex. Based on differences in accessible surface areas between GPR30 and its ligand, the residues in the interaction zone were identified. The cyclopeptide is stabilized in the active site by forming a network of hydrogen bonds between Glu, Thr, (1)Pro(OH) and GLn residues of the ligand and Arg-259, Cys-271, Asn-316, Asn-320 and Tyr-324 of the G-protein. Moreover, the study of the electrostatic surface potential on the GPR30 receptor shows that the active site is more positively charged than the other sites. Our modeling indicates a plausible interaction of the cyclopeptide with the seven transmembrane GPR30 protein. This may have profound implications for the treatment of breast cancer.

DNA bending and sugar switching.

Abstract Using high frequency antiphase NMR spectroscopy and computer simulations of the antiphase spectra, we studied the equlibria in the sugar conformations in the DNA duplex 11-mer containing the AAA tract surrounded by cytosines. We demonstrate that at the 3'-end of the A-tract, the sugar switches from the common S-conformer (B-like form) to the N-conformer (A-like form) with the probability of 50-60%, thus creating a purine-pyrimidine step with heteronomous characteristics. The presence of this local B-A junction in one strand leads to compression of the interphosphate distance in this strand. We calculate the effect of this sugar switch on the helical parameters that are related to DNA bending. Finally, we suggest that the heteronomous deformation present in the A(n)C motif of unbound DNA duplexes might be the initial recognition site for proteins which bind to such junctions; and that in A(n)C stretches, DNA bending is a complicated dynamic process, i.e., locally noncanonical N conformers of the sugar phosphate backbone mix in with the B-like S conformers leading to bending.

DNA bending and sequence-dependent backbone conformation NMR and computer experiments.

Although DNA bending plays a crucial role in several biological processes, very little is known experimentally about the relationship between sugar phosphate conformation and sequence directed bending. In this paper, we determine the coupling constants for a nonself-complementary 11-mer A-tract DNA duplex from 2D NMR experiments and along each chain of the duplex, we report the sugar pucker, torsional preferences and conformational averaging about the C3'-O3', C4'-C5' and C5'-O5' bonds for each nucleotide. The A-tract exists as an equilibrium blend of canonical B-form and noncanonical B-form in which the exocyclic C4'-C5' bond is in trans conformation as in the original Watson-Crick model [Crick, F.H.C. & Watson, J.D. (1954) Proc. Roy. Soc. (London), A223, 80-96]. The trans conformation at the C4'-C5' can increase the interphosphate distance and lead to local unwinding of the duplex and rolling of the base pair into the major groove. This will create a kink or hinge. At the 3'-end of the A-tract in the purine-thymine step, the duplex is compressed by the presence of a junction between A and B forms of DNA exclusively in one strand, with consequent reduction of the phosphate-phosphate distance. The coupling constant data seriously disagree with the A-tract DNA bending model of Crothers [Koo, H.-S., Wu, H.-M. & Crothers, D.M. (1986) Nature 320, 501-506], but is in agreement with the finding of Leroy et al. [Leroy, J.-L., Charretier, E., Kochoyan, M. & Gueron, M. (1988) Biochemistry 27, 8894-8898] that the structure that drives bending in the A-tract is locally different from B-DNA. Structural distortions are extremely localized with little or no propagation. It is likely that transcription factor proteins recognize these preexisting deformations in the free DNA itself and mold it into the matrix of the protein.

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.

GC rich DNA oligonucleotides with narrow minor groove width.

Investigation of the width of the minor groove using 500 MHz NMR spectroscopy in three closely related 11-mer B-DNA duplexes shows that the minor groove is narrow in a GC rich oligonucleotide, and that a narrow minor groove is not something endemic to DNAs with persistent repetitions of adenine nucleotides (A-tract DNA). The width of the groove is dictated by local sequence contexts and independent of neighboring A-tract DNA.

Modeling of a possible conformational change associated with the catalytic mechanism in the hammerhead ribozyme.

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water.

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

Decreased interstrand H2-H1' distance in the GC-rich part of the duplex d(CCTCAAACTCC).d(GGAGTTTGAGG) in solution at low temperature: proton nuclear magnetic resonance investigation.

The non-self-complementary undecadeoxyribonucleotide duplex d(CCTCAAACTCC).d(GGAGTTTGAGG) was studied by one- and two-dimensional NMR methods in solution at low and room temperatures. The width of the minor groove of the duplex was determined on the basis of the NOE's between adenine's H2 protons and H1' protons from the complementary strand. In agreement with the previous reports, we found that the A3.T3 block forms a structure with a narrow minor groove at 5 degrees C, with the H2-H1' interstrand distance decreasing in the 5'-to-3' direction along the strand of adenines. Surprisingly, this distance is still short in the GC-rich part of the duplex downstream from the A-tract. This finding is interpreted in terms of pronounced buckle angles in the oligo(purine).oligo(pyrimidine) blocks, which diminish the H2-H1' interstrand distances. Both CA(n)- and AnC- junctions have distinct patterns of the proton chemical shifts, which suggests that both junctions may have some specific conformations in solution. Also, we report the temperature-driven changes in proton chemical shifts, which are significant in all parts of the duplex, except the 5'-ends of both strands. The structural interpretation for these changes is proposed, on the basis of the following notion: at low temperature the narrow minor groove is formed between the central A3.T3 trimer and the 3'-ends of both DNA strands, while the 5'-ends remain relatively exposed to the solvent.

NMR study of self-paired parallel duplex of d(AAAAACCCCC) in solution.

The oligonucleotide d(A5C5) in solution forms a parallel self-duplex at neutral and low pH values. H2O NMR spectra at pH 5.1 indicate the presence of five imino resonances at lower temperatures; and the structure is stable up to 60 degrees C. These signals can arise only from the hemiprotonated C+.C pairs [Westhof, E. and Sundaralingham, M. (1980) Biochemistry 77, 1852-1856; Westhof, E. and Sundaralingham, M. (1980) J. Mol. Biol. 142, 331-361] and constitute the first direct observation of C+.C hemiprotonated pairs in solution. The cross peaks from H1's and more than five distinct AH8's in 500 MHz 1H 2D-NOESY spectra indicate that there are two conformationally different and energetically similar A-tracts. There is good qualitative agreement between NOESY data and two theoretically derived structures in which A-tracts are reverse Watson-Crick and reverse Hoogsteen base-paired, respectively.

Tetraplex formation of d(GGGGGTTTTT): 1H NMR study in solution.

The oligonucleotide d(G5T5) can in principle form a fully matched duplex with G.T pairing and/or a tetraplex. Non-denaturing gel electrophoresis, circular dichroism and NMR experiments show that the tetraplex is exclusively formed by this oligomer in solution. In the presence of its complementary strand d(A5C5) at low temperature, d(G5T5) forms the tetraplex over the normally expected Watson-Crick duplex. However, when d(G5T5) and d(A5C5) are mixed together in equimolar amounts and heated for several minutes at 85 degrees C, and then allowed to cool, the product was essentially the Watson-Crick duplex. The lack of resolution in the 500 MHz 1H NMR spectra and the presence of extensive spin diffusion do not allow us to derive a quantitative structure for the tetraplex from the NMR data. However, we find good qualitative agreement between the NOESY and MINSY data and a theoretically derived stereochemically sound structure in which the G's and T's are part of a parallel tetraplex.

Systematic study of nuclear Overhauser effects vis-à-vis local helical parameters, sugar puckers, and glycosidic torsions in B DNA: insensitivity of NOE to local transitions in B DNA oligonucleotides due to internal structural compensations.

A method has been developed to solve structures of DNA oligomers in solution from the experimental NOE data. The method is a combination of two approaches: (1) full matrix NOESY simulations and (2) conformational calculations of DNA double helix based on generalized helical parameters. The process of the refinement of a solution structure does not involve NMR-derived interproton distance constraints; rather it consists of a direct fitting of a structure to the experimental NOE data, a weighted sum of energy, and R factor being under minimization. A helical parameters-based generation of DNA forms makes it possible to organize the search for the optimal structure more effectively, systematically varying starting conformations. The method has been used to calculate a structure for the self-complementary DNA hexamer GGATCC, which is consistent with the available experimental data. The structure belongs to the B family of forms, although the local structural heterogeneity is very strong. Sugar puckers vary from O4'-exo to C3'-exo; helical steps are open with different magnitudes toward the minor groove. Next, we have addressed the question of how uniquely the structure is defined by the existing NMR data. Different structural parameters have been systematically varied, and their effect on individual NOE's and the R factor has been studied. Two energetically conjugated parameters, sugar puckers and glycosidic angles, can be determined very reliably, because of the strong dependences of the intraresidue H6/H8 to H2'/H2''/H3' NOE's. In contrast, the local helical conformation of DNA and the geometry of base pairs proved to be underdetermined by the existing NOE information, because the effect of any helical parameter on interproton distances can be compensated by the concerted changes in other parameters.

In search of a Hoogsteen base paired DNA duplex in aqueous solution.

When the oligodeoxynucleotides d(A)6 and d(T)6 are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC in equilibrium with H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)6. We show that (i) addition of extra d(A)6 does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix-drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, 31P NMR spectra, 1H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded. Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.