Conformational analysis of the 3'-5'-cyclic dinucleotide d less than pApA greater than by means of molecular mechanics

The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Dinucleoside monophosphate having a high anti conformation: the crystal structure of 8,2'-S-cycloadenylyl-(3'-5')-8,2'-S-cycloadenosine hydrochloride

The crystal and molecular structure of 8,2'-S-cycloadenylyl-(3'-5')-8,2'-S-cycloadenosine (AspAs) hydrochloride has been determined by X-ray method. The conformation of two independent AspAs molecules found in an asymmetric unit are almost identical to each other. The torsion angles concerning the sugar-phosphate backbone are different from those in crystalline dinucleoside monophosphates so far determined by X-rays. Both AspAs molecules are in the sharp bend conformations, i.e. each rotation around P-O bond (omega', omega) is (g-, t) rather than the preferred (g-, g-) or (g+, g+) conformation. There is no intramolecular base stacking or base-pairing but the intermolecular base stacking was found.     

Conformational Analysis of the 3′-5′-Cyclic Dinucleotide d<pApA> by Means of Molecular Mechanics

Abstract

The conformational properties of the cyclic dinucleotide d<(pApA)> were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: α⁺, β¹, γ⁺, ?¹ and ζ⁺. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3′ atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of γ⁺ to γ^?. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180° change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA are found in the dataset obtained for cyclic d<(pApA)>. It turns out that in the available examples, 180° changes in the backbone direction are localized between two adjacent nucleotides.     

Parallel DNA double helices. II. Conformational analysis of regular helices having a second order axis of symmetry

Conformational analysis of double helices of DNA with parallel arranged sugar-phosphate chains connected by twofold symmetry has been performed. Homopolymers poly(dA).poly(dA), poly(dC).poly(dC), poly(dG).poly(dG) and poly(dT).poly(dT) were studied. For each of the homopolymers all variants of H-bond pairing were checked. The maps of closing of sugar-phosphate backbone were previously computed. By the optimization of potential energy the dihedral angles and helix parameters of relatively stable conformations of parallel stranded polynucleotides were calculated. The dependence of conformational energy on the nucleic base character and the base pair type were studied. Two main conformational regions for favourable "parallel" helix of polynucleotides were found. The former of these two regions coincide with the region of typical conformational parameters of B-DNA. On an average the conformational energy of "parallel" DNA is close to the energy of canonic "antiparallel" B-DNA.     

Secondary structure of the hybrid poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and stereochemical modelling within the constraints of NOE data

One-dimensional nuclear Overhauser effect (NOE) in nuclear magnetic resonance spectroscopy along with stereochemically sound model building was employed to derive the structure of the hybrid poly(rA).poly(dT) in solution. Extremely strong NOE was observed at AH2' when AH8 was presaturated; strong NOEs were observed at TH2'TH2' when TH6 was presaturated; in addition the observed NOEs at TH2' and TH2' were nearly equal when TH6 was presaturated. There was no NOE transfer to AH3' from AH8 ruling out the possibility of (C-3'-endo, low anti chi approximately equal to 200 degrees to 220 degrees) conformation for the A residues. The observed NOE data suggest that the nucleotidyl units in both rA and dT strands have equivalent conformations: C-2'-endo/C-1'-exo, anti chi approximately equal to 240 degrees to 260 degrees. Such a nucleotide geometry for rA/dT is consistent with a right-handed B-DNA model for poly(rA).poly(dT) in solution in which the rA and dT strands are conformationally equivalent. Molecular models were generated for poly(rA).poly(dT) in the B-form based upon the geometrical constraints as obtained from the NOE data. Incorporation of (C-2'-endo pucker, chi congruent to 240 degrees to 260 degrees) into the classical B-form resulted in severe close contacts in the rA chain. By introducing base-displacement, tilt and twist along with concomitant changes in the backbone torsion angles, we were able to generate a B-form for the hybrid poly(rA).poly(dT) fully consistent with the observed NOE data. In the derived model the sugar pucker is C-1'-exo, a minor variant of C-2'-endo and the sugar base torsion is 243 degrees, the remaining torsion angles being: epsilon = 198 degrees, xi = 260 degrees, alpha = 286 degrees, beta = 161 degrees and gamma = 72 degrees; this structure is free of any steric compression and indicates that it is not necessary to switch to C-3'-endo pucker for rA residues in order to accommodate the 2'-OH group. The structure that we have proposed for the polynucleotide RNA-DNA hybrid in solution is in complete agreement with that proposed for a hexamer hybrid in solution from NOE data and is inconsistent with the heteronomous model proposed for the fibrous state.     

The solution structure of the intramolecular photoproduct of d(TpA) derived with the use of NMR and a combination of distance geometry and molecular dynamics.

One and two dimensional NMR techniques have been used together with molecular modelling to obtain the solution structure for the photoproduct d(TpA)*. The NMR data confirm that the cyclobutane linkage is formed between the bonds thymine C6-C5 and adenine C5-C6. The 2D NOE data are used as constraints in a distance geometry calculation. The structures obtained show a trans-syn cyclobutane linkage and the glycosidic angles are SYN and ANTI for thymidine and deoxyadenosine, respectively. The coupling constant data are used to check the backbone torsion angles of the obtained structures. Typical torsion angles are a gamma+ and beta t for the deoxyadenosine residue. A free molecular dynamics simulation of a trans-syn d(TpA) photoproduct confirmed all these structural characteristics.     

Molecular structure of cyclic deoxydiadenylic acid at atomic resolution

The molecular structure of a small cyclic nucleotide, cyclic deoxydiadenylic acid, has been determined by single-crystal X-ray diffraction analysis and refined to an R factor of 7.8% at 1.0-A resolution. The crystals are in the monoclinic space group C2 with unit cell dimensions of a = 24.511 (3) A, b = 24.785 (3) A, c = 13.743 (3) A, and beta = 94.02 (2) degrees. The structure was solved by the direct methods program SHELXS-86. There are 2 independent cyclic d(ApAp) molecules, 2 hydrated magnesium ions, and 26 water molecules in the asymmetric unit of the unit cell. The two cyclic d(ApAp) molecules have similar conformations within their 12-membered sugar-phosphate backbone ring, but they have quite different appearances due to the different glycosyl torsion angles that make one molecule more compact and the other extended and open. Three of the four deoxyribose rings are in the less common C3'-endo conformation. All four phosphate groups have their phosphodiester torsion angles alpha/zeta in the gauche(+)/gauche(+) conformation. One of the cyclic d(ApAp) molecules associates with another symmetry-related molecule to form a self-intercalated dimer that is a stable structure in solution, as observed in NMR studies. Many interesting intermolecular interactions, including base-base stacking, ribose-base stacking, base pairing, base-phosphate hydrogen bonding, and metal ion-phosphate interactions, are found in the crystal lattice. This structure may be relevant for understanding the conformational potentiality of an endogenous biological regulator of cellulose synthesis, cyclic (GpGp).     

Sequence-dependent conformation of an A-DNA double helix. The crystal structure of the octamer d(G-G-T-A-T-A-C-C)

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1.8 and 2.25 A, respectively. The molecules adopt and A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.     

Lipid conformation in crystalline bilayers and in crystals of transmembrane proteins

Dihedral torsion angles evaluated for the phospholipid molecules resolved in the X-ray structures of transmembrane proteins in crystals are compared with those of phospholipids in bilayer crystals, and with the phospholipid conformations in fluid membranes. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1-C2 rotamers found invariably in phospholipid bilayer crystals. Lipid headgroup conformations in protein crystals also do not conform solely to the bent-down conformation, with gauche-gauche configuration of the phospho-diester, that is characteristic of phospholipid bilayer membranes. This suggests that the lipids that are resolved in crystals of membrane proteins are not representative of the entire lipid-protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from energetically disallowed skew conformations. This indicates a configurational heterogeneity in the lipids at a single binding site: eclipsed conformations occur also in some glycerol backbone torsion angles and C-C torsion angles in the lipid headgroups. Stereochemical violations in the protein-bound lipids are evidenced by one-third of the ester carboxyl groups in non-planar configurations, and certain of the carboxyls in the cis configuration. Some of the lipid structures in protein crystals have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in structures of the phytanyl chains associated with bacteriorhodopsin crystals are in the incorrect S-configuration.     

Four-stranded DNA. Conformational analysis of regular spirals of poly(dT).poly(dA).poly(dA).poly(dT) with different base binding variations

Conformational analysis of four stranded DNA helices poly(dT).poly(dA).poly(dA).poly(dT) with parallel arrangement of the identical sugar-phosphate chains connected by twofold symmetry has been performed. All possible models of symmetrical base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of four stranded polynucleotides were calculated. The dependences of conformational energy on the base complex structure and mutual orientation of the poly(dA).and poly(dT) chains were studied. Possible biological functions of four stranded helices are discussed.     

Crystal and solution structures of the B-DNA dodecamer d(CGCAAATTTGCG) probed by Raman spectroscopy: heterogeneity in the crystal structure does not persist in the solution structure

The self-complementary dodecamer d(CGCAAATTTGCG) crystallizes as a double helix of the B form and manifests a Raman spectrum with features not observed in Raman spectra of either DNA solutions or wet DNA fibers. A number of Raman bands are assigned to specific nucleoside sugar and phosphodiester conformations associated with this model B-DNA crystal structure. The Raman bands proposed as markers of the crystalline B-DNA structure are compared and contrasted with previously proposed markers of Z-DNA and A-DNA crystals. The results indicate that the three canonical forms of DNA can be readily distinguished by Raman spectroscopy. However, unlike Z-DNA and A-DNA, which retain their characteristic Raman fingerprints in aqueous solution, the B-DNA Raman spectrum is not completely conserved between crystal and solution states. The Raman spectra reveal greater heterogeneity of nucleoside conformations (sugar puckers) in the DNA molecules of the crystal structure than in those of the solution structure. The results are consistent with conversion of one-third of the dG residues from the C2'-endo/anti conformation in the solution structure to another conformation, deduced to be C1'-exo/anti, in the crystal. The dodecamer crystal also exhibits unusually broad Raman bands at 790 and 820 cm-1, associated with the geometry of the phosphodiester backbone and indicating a wider range of (alpha, zeta) backbone torsion angles in the crystal than in the solution structure. The results suggest that backbone torsion angles in the CGC and GCG sequences, which flank the central AAATTT sequence, are significantly different for crystal and solution structures, the former containing the greater diversity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleic acid model building: the multiple backbone solutions associated with a given base morphology

A constrained model building procedure is used to generate nucleic acid structures of the familiar A-, B-, and Z-DNA duplexes. Attention is focused upon the multiple structural solutions associated with the arrangements of nucleic acid base pairs rather than the optimum sugar-phosphate structure. The glycosyl (chi) and sugar torsions (both the ring puckering and the exocyclic C5'-C4' (psi) torsion) are treated as independent variables and the resulting O3'...O5' distances are used as closure determinants. When such distances conform to the known geometry of phosphate chemical bonding, an intervening phosphorus atom with correct C-O-P valence angles can be located. Four sequential torsion angles--phi', omega', omega and phi--about the C3'-O3'-P-O5'-C5' bonds are then obtained as dependent variables. The resulting structures are categorized in terms of conformation, ranked in potential energy, and analyzed for torsional correlations. The numerical results are quite interesting with implications regarding nucleic acid models constructed to fit less than ideal experimental data. The multiple solutions to the problem are useful for comprehending the conformational complexities of the local sugar-phosphate backbone and for understanding the transitions between different helical forms. According to these studies, unique characterization of a nucleic acid duplex involves more than the determination of its base pair morphology, its sugar puckering preferences, or its groove binding features.     

Structure of a triple helical DNA with a triplex-duplex junction

Extended purine sequences on a DNA strand can lead to the formation of triplex DNA in which the third strand runs parallel to the purine strand. Triplex DNA structures have been proposed to play a role in gene expression and recombination and also have potential application as antisense inhibitors of gene expression. Triplex structures have been studied in solution by NMR, but have hitherto resisted attempts at crystallization. Here, we report a novel design of DNA sequences, which allows the first crystallographic study of DNA segment containing triplexes and its junction with a duplex. In the 1.8 A resolution structure, the sugar-phosphate backbone of the third strand is parallel to the purine-rich strand. The bases of the third strand associate with the Watson and Crick duplex via Hoogsteen-type interactions, resulting in three consecutive C(+).GC, BU.ABU (BU = 5-bromouracil), and C(+).GC triplets. The overall conformation of the DNA triplex has some similarity to the B-form, but is distinct from both A- and B-forms. There are large changes in the phosphate backbone torsion angles (particularly gamma) of the purine strand, probably due to the electrostatic interactions between the phosphate groups and the protonated cytosine. These changes narrow the minor groove width of the purine-Hoogsteen strands and may represent sequence-specific structural variations of the DNA triplex.     

Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR

The cytoplasmic tail of the amyloid precursor protein (APPc) interacts with several cellular factors implicated in intracellular signaling or proteolytic production of amyloid beta peptide found in senile plaques of Alzheimer's disease patients. APPc contains two threonine residues (654 and 668 relative to APP695, or 6 and 20 relative to APPc) and a serine residue (655 or 7, respectively) that are known to be phosphorylated in vivo and may play regulatory roles in these events. We show by solution NMR spectroscopy of a 49 residue cytoplasmic tail peptide (APP-C) that in all three cases, phosphorylation induces changes in backbone dihedral angles that can be attributed to formation of local hydrogen bonds between the phosphate group and nearby amide protons. Phosphorylation of S7 also induces chemical shift changes in the hydrophobic cluster (residues I8-V13), indicating additional medium-range effects. The most pronounced changes occur upon phosphorylation of T20, a neuron-specific phosphorylation site, where the N-terminal helix capping box previously characterized for this region is altered. Characterization of torsion angles and transient hydrogen bonds indicates that prolyl isomerization of the pThr-Pro peptide bond results from both destabilization of the N-terminal helix capping box and stabilization of the cis isomer by transient hydrogen bonds. The significant population of the cis isomer (9 %) present after phosphorylation of T20 suggests a potential role of selective recognition of cis versus trans isomers in response to phosphorylation of APP. Together, these structural changes indicate that phosphorylation may act as a conformational switch in the cytoplasmic tail of APP to alter specificity and affinity of binding to cytosolic partners, particularly in response to the abnormal phosphorylation events associated with Alzheimer's disease.     

DNA with adenine tracts contains poly(dA).poly(dT) conformational features in solution.

The conformation of DNA's with adenine-thymine tracts exhibiting retardation in electrophoretic migration and considered as curved were investigated in solution by CD and RAMAN spectroscopy. The following curved multimers with adenine tracts but of different flanking sequences d(CA5TGCC)n, d(TCTCTA6TATATA5)n, d(GA4T4C)n yield CD spectroscopic features indicating a non-B structure of the dA.dT tract with similarities to polyd(A).polyd(T). We suggest that adenine-thymine bases in these multimers contain some of the distinctive conformational features of poly(A).polyd(T) probably with large propeller twist found by NMR (Behling and Kearns, 1987) and by X-ray diffraction on oligonucleotides containing a tract of adenines (Nelson et al. 1987, Coll et al; 1987; DiGabriele et al. 1989). Some elements of distinctive CD features of the contiguous adenines run are also observed in the straight multi-9-mer d(CA5GCC)n which lacks in-phase relation to the helical repeat. Despite the presence of the TpA step in the straight multimer d(GT4A4)n, the altered dA.dT conformation is not completely destroyed. Interruption of adenine tract by a guanine in d(CAAGAATGCC)n leads to a B-like conformation and to a normal electrophoretic mobility. The Raman spectra reveal a rearrangement of the sugar-phosphate backbone of dA.dT tract in the multimer d(CA5TGCC)n with respect to that of polydA.polydT. This is reflected in the presence of an unique Raman band associated to C2'-endo sugar with a predominant contribution of C1'-exo puckering which is exhibited by the multimer whereas two distinct Raman bands characterize poly(dA).poly(dT) backbone conformation.     

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.     

The structure of the double-stranded RNA pentamer 5'(CACAG) . 5'(CUGUG) determined by nuclear Overhauser enhancement measurements: interproton distance determination and structure refinement on the basis of X-ray coordinates

The structure in solution of the duplex RNA pentamer 5'(CACAG) . 5'(CUGUG), comprising the stem of the T psi C loop of yeast tRNAPhe, has been investigated by means of one- and two-dimensional nuclear Overhauser enhancement measurements. All non-exchangeable base and sugar proton resonances with the exception of the H5'/H5" sugar resonances are assigned in a sequential manner. From the relative intensities of the cross-peaks obtained in the pure-phase absorption two-dimensional nuclear Overhauser enhancement spectra at several mixing times, it is deduced that the RNA pentamer adopts an A-type conformation in solution. Cross-relaxation rates and interproton distances are determined from the time dependence of the nuclear Overhauser effects, principally by one-dimensional measurements. The structure of the RNA pentamer is then refined by restrained least-squares minimization on the basis of both distance and planarity restraints using fibre diffraction data as an initial model. The refined structure of the RNA pentamer is of the A type but exhibits local structural variations in glycosidic bond and backbone torsion angles as well as in propeller twist, base roll and base tilt angles.