Pseudorotational Angle Research Articles

Extension of the Karplus Relationship for NMR Spin−Spin Coupling Constants to Nonplanar Ring Systems: Pseudorotation of Cyclopentane

A new Karplus equation for pseudorotating five-membered rings is derived by expanding calculated NMR spin−spin coupling constants (SSCCs) J as a function J(q,φ) of the puckering amplitude q and the pseudorotational phase angle φ. The approach was tested for cyclopentane but is equally applicable to ribose sugars and other biochemically interesting five-membered rings. It is based on the calculation of the conformational potential V(q,φ), which in the case of cyclopentane was determined at the MBPT(2)/cc-pVTZ level of theory. Cyclopentane is a free pseudorotor (barriers ΔE and ΔH ≤ 0.01 kcal/mol) with a puckering amplitude q = 0.43 Å and a barrier to inversion ΔH = 5.1 kcal/mol, in perfect agreement with experimental data. The SSCCs of cyclopentane were calculated at MBPT(2)/cc-pVTZ geometries by use of coupled perturbed density functional theory (CP-DFT) with the B3LYP functional and a (9s,5p,1d/5s,1p)[6s,4p,1d/3s,1p] basis set. In addition, coupled-cluster singles and doubles (CCSD) calculations were carried out to verify the CP-DFT results. All geometrical parameters and the 10 SSCCs of cyclopentane are determined as functions of the phase angle φ and averaged to give 〈nJ〉 values that can be compared with experimental data. The following SSCCs (in hertz) were obtained at CP-DFT/B3LYP/[6s,4p,1d/3s,1p]: 〈1J(CC)〉 = 34.0; 〈1J(CH)〉 = 127.6, exp 128.2; 〈2J(CCC)〉 = 2.3, exp (+)2.8; 〈2J(CCH)〉 = −2.6, exp (−)3.0; 〈2J(HCH)〉 = −12.4, exp (−)12.4; 〈3J(CCCH)〉 = 3.9; 〈3J(HCCH, cis)〉 = 7.7, exp 7.7; 〈3J(HCCH, trans)〉 = 5.6, exp 6.3; 〈4J(HCCCH, cis)〉 = 0.1; 〈4J(HCCCH, trans)〉 = −0.6. Magnitude and trends in calculated SSCCs are dominated by the Fermi contact term [with the exception of 1J(CC)]. A new way of determining puckering amplitude and pseudorotational angle by a combination of measured and calculated SSCCs is presented.

Vibrational Calculations for the HD2 First-Excited Electronic State Using a Coordinate-Transformation Technique

We report calculations of the vibrational spectrum of HD2 in its first-excited electronic state by using a coordinate-transformation technique. The implications of the geometric phase (GP) effect in the spectrum have been investigated. The results show that the lowest 45 vibrational levels calculated using the traditional Longuet−Higgins phase (φ/2, where φ is the pseudorotation angle) are in good agreement with those obtained by using a single-surface generalized Born−Oppenheimer equation previously reported by the authors.

Use of Oligodeoxyribonucleotides with Conformationally Constrained Abasic Sugar Targets To Probe the Mechanism of Base Flipping by HhaI DNA (Cytosine C5)-methyltransferase

X-ray crystallographic studies of HhaI DNA (cytosine-C5)-methyltransferase (M.HhaI) covalently linked to methylated 5-fluorocytosine in DNA provided the first direct evidence that the cytosine residue targeted for methylation was “flipped” out of the helix during the transfer reaction. Subsequent studies indicated that removal of the target cytosine base, i.e., introduction of an abasic site, enhanced binding of M.HhaI to DNA and that the conformation of the sugar−phosphate backbone at the abasic site in the resultant complexes was the same as that of the sugar attached to a “flipped” cytosine. In the present study, pseudorotationally constrained sugar analogues, based on bicyclo[3.1.0]hexane templates, were placed in DNA duplexes as abasic target sites in the M.HhaI recognition sequence. Biochemical studies demonstrate that binding affinity of M.HhaI for abasic sites increases when the abasic target sugar analogue is constrained to the south conformation and decreases when it is constrained to the north conformation. In native gel-shift assays, M.HhaI exhibits a “closed” conformation when bound to the abasic south or abasic furanose analogues, whereas an “open” conformation predominates with the abasic north analogue. A structural understanding of these results was obtained via molecular dynamics simulations of the DNA duplex alone and in ternary complex with M.HhaI and cofactor, along with quantum mechanical calculations on model compounds representative of the abasic and modified sugars. Binding affinities are shown to be related to the ability of the abasic sugar analogues to spontaneously flip out of the DNA duplex. Enhanced binding of the abasic south analogue is suggested to be due to its increased capacity for sampling the experimentally observed conformation of the DNA target site in the M.HhaI ternary complex. Decreased binding of the north analogue is due to decreased flexibility of the phosphodiester backbone associated with a north pseudorotation angle, thereby inhibiting spontaneous flipping of the sugar moiety out of the DNA duplex. Spontaneous flipping of the sugar moiety out of the DNA duplex is also suggested to facilitate formation of a “closed” complex between M.HhaI and DNA whereas partial or no flipping favors the “open” conformation. These results show that introduction of structural constraints into DNA that induce enhanced sampling of protein-bound conformations facilitate DNA−protein binding. Implications of the present results with respect to the mechanism of base flipping in the M.HhaI catalytic cycle are discussed.

Crystal Structure and Molecular Conformation of 4-Thiouracil-2′-Trifluorothioacetamide -3′, 5′-Diacetyl-β-D-Riboside

4-thiouracil-2′-trifluorothioacetamide-3′, 5′-diacetyl-β-D-riboside is one of the modified thiouracil analogs synthesized in our institute. The determination of the crystal and molecular structure of this compound was carried out with a view to study the conformation of the molecule in the solid state as well as to investigate the conformations of the trifluoroacetamide and the acetyl substituents of the ribose and their effects on the conformation of the ribose ring. Crystals of 4-thiouracil-2′-trifluorothioacetamide-3′,5′- diacetyl-β-D-riboside are orthorhombic, space group P21 21 21, with cell dimensions a= 15.351 (2), b= 15.535 (1), c= 8.307 (1) Å, V=1981.0 (7) Å3, Z=4, Dm= 1.53, Dc=1.527 g/c.c. and μ=30.1cm -1. The structure was determined using CuKα (λ, =1.5418 Å) at a temperature T of 297K, with 2333 reflections, which were collected on a Enraf-Nonius CAD-4 diffactometer, out of which 2249 (I ≥20) were considered observed. The structure was determined by direct methods using MULTAN and refined by full matrix least squares method to a final reliability factor of 0.054 and a weighted R factor of 0.079. The nucleoside is in the anti conformation [XCN =51.4 (5)°], the ribose has the unusual C (2′) endo -C (1′) exo (2T1), and a g+ conformation [ψ=47.5 (4)] across C(4′)-C(5′) bond. The pseudorotation angle P is 152.8 (4) ° and the amplitude of pucker τm of 42.7 (3)°. The average C-F bond distance is 1.308 Å. There is no base pairing and the typical base-base hydrogen bonded interactions are not present in this structure. On the other hand, a hydrogen bonded dimer is formed involving C(3′) - H(3′)… O (2) and N(3) -H (N3) … O (Al) hydrogen bonds joining the base, ribose ring and the acetyl group. The trend towards longer exocyclic bonds at the acetyl centers in compounds with strongly electronegative aglycones, is also exhibited in this compound, with C(3′)-O(3′) and C(5′)-0(5′) being much longer than C(1′)-O(4′). The acetyl groups also take part in C-H…O hydrogen bonding with the acetyl oxygen atom OA2.

Multiple conformations of an intercalated (-)-(7S,8R,9S, 10R)-N6-[10-(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence.

The structure of the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct at A7 of 5'-d(CGGACAAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', derived from trans addition of the exocyclic N6-amino group of dA to (-)-(7S,8R,9R, 10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-DE2], was determined using molecular dynamics simulations restrained by 532 NOEs from 1H NMR. This was named the SRSR(61,3) adduct, derived from the N-rasprotooncogene at and adjacent to the nucleotides encoding amino acid 61 (underlined) of the p21 gene product. The solution structure of this adduct was best described as a mixture of two conformations in rapid equilibrium on the NMR time scale. The two populations differed in the pseudorotation angle of the sugar ring for the 5'-neighboring base A6, as determined from scalar coupling data. One population, estimated to be present at 53%, had the A6 deoxyribose in the C2'-endo conformation, while in the second conformation the A6 deoxyribose was in the C3'-endo conformation. NOEs between C5, A6, and SRSRA7 were either disrupted or weakened, as were those in the complementary strand between C15, T16, and T17. Major groove NOEs were observed between the benzo[a]pyrene aromatic protons, H1, H2, H3, H4, H5, and H6, and T16 CH3. Minor groove NOEs were observed between H1, H2, and H3 of benzo[a]pyrene and T16 H1' and H2' and T17 H1' and H2'. The benzo[a]pyrene protons H10, H11, and H12 showed NOEs to A6 H1', H2', and H2". The chemical shifts of the pyrenyl moiety were dispersed over a 1.9 ppm range. Upfield chemical shifts of 2.4 ppm for T16 N3H, 1.1 ppm for T17 N3H, 1.3 and 1.0 ppm for T16 H6 and CH3, 0.85 ppm for T16 H1', and 0.80 and 0.90 ppm for C15 H2' and H2" were observed. These observations were consistent with intercalation of the pyrenyl moiety toward the 5' direction of SRSRA7. The results were compared to the isomeric SRSR(61,2) adduct [I. S. Zegar, S. J. Kim, T. N. Johansen, P. J. Horton, C. M. Harris, T. M. Harris, and M. P. Stone (1996) Biochemistry 35, 6212-6224] and revealed the role of DNA sequence in modulating the conformation of this benzo[a]pyrene adduct.

Molecular Structure of Two Crystal Forms of Cyclic Triadenylic Acid at 1Å Resolution

The three dimensional structures of cyclic deoxytriadenylic acid, c-d(ApApAp), from two different trigonal crystal forms (space groups P3 and R32) have been determined by x-ray diffraction analysis at 1Å resolution. Both structures were solved by direct methods and refined by anisotropic least squares refinement to R-factors of 0.109 and 0.137 for the P3 and R32 forms, respectively. In both crystal forms, each of the two independent c-d(ApApAp) molecules sits on the crystallographic 3-fold axis. All four independent c-d(ApApAp) molecules have similar backbone conformations. The deoxyriboses are in the S-type pucker with pseudorotation angles ranging from 156.7° to 168.6° and the bases have anti glycosyl torsion angles (χ falling in two ranges, one at −104.3° and the other ranging from −141.0° to −143.8°). In the R32 form, a hexahydrated cobalt(II) ion is found to coordinate through bridging water molecules to NI, N3, and N7 atoms of three adjacent adenines and oxygen atoms of phosphates. Comparison with other structures of cyclic oligonucleotides indicates that the sugar adopts N-type pucker in cyclic dinucleotides and S-type pucker in cyclic trinucleotides, regardless whether the sugar is a ribose or a deoxyribose.

NMR Conformation of (−)-β-d-Aristeromycin and Its 2‘-Deoxy and 3‘-Deoxy Counterparts in Aqueous Solution

The solution conformations of aristeromycin (1), 2‘-deoxyaristeromycin (2), and 3‘-deoxyaristeromycin (3) have been determined from an integrated analysis of X-ray (for 1 only), NMR data (i.e., 3JHH coupling constants), and ab initio calculations. One-dimensional NOE difference experiments showed that the adenin-9-yl ring in 1 and 2 is involved in a ∼50% syn ⇄ ∼50% anti equilibrium around the C-glycosyl torsion angle, whereas an anti orientation (χ = −113°) is found in the X-ray crystal structure of 1. The preferred conformation around the γ torsion angle is γt both in solution for 1−3 and in the solid state (for 1 only). The plots of energy as a function of the phase angle of pseudorotation (Figure 2) for the structures optimized by ab initio calculations (HF/3-21G*) show that there are two major wells of low energy conformers for 1−3, supporting the two-state North-type ⇄ South/West-type equilibrium of the constituent cyclopentane rings in 1−3. The ab initio calculations suggested that the South/West-type conformers are more stable than the North-type forms for 1 [ΔE (120° < P < 240°) − (330° < P < 30°) ∼10 kcal/mol], for 2 [ΔE (210° < P < 240°) − (330° < P < 60°) ∼ 4 kcal/mol] and for 3 [ΔE (P = 240°) − (330° < P < 0°) ∼ 6 kcal/mol]. Newly developed A and B sets of parameters correlating the H−C−C−H torsions to the endocyclic torsions based on the ab initio optimised structures of 1−3 have been subsequently used to interpret the time-averaged 3JHH couplings using the program PSEUROT. The discrepancy found between the X-ray crystal structure (P = 89°, Ψm = 41°) of aristeromycin (1) and its structure calculated by NMR-PSEUROT conformational analysis (35° < P [ T − T] < 65°, 35° < Ψm < 45°) ⇄ (128° < P [1E] < 131°, 34° < Ψm < 36°) based on observed 3JHH couplings in aqueous solution, as well as the relatively high error in the NMR-PSEUROT analyses for 1−3 [ΔJmax ≤ 1.6 Hz (i.e., maximal difference between experimental and PSEUROT-calculated 3JHH) and root mean square (rms) error ≤ 0.7 Hz] prompted us to reparametrize the Karplus equation implemented in the PSEUROT program by using torsion angles derived from solid-state geometries of conformationally constrained nucleosides and their corresponding experimental 3JHH. The precision of our reparametrized Karplus-type equation (rms error = 0.40 Hz) became comparable to that expected for the standard Haasnoot−Altona Karplus (0.48 Hz) equation. The results of the PSEUROT analyses performed with the standard Haasnoot−Altona Karplus equation are also very comparable in terms of geometry with those based on our reparametrized equation (eq 4). Both series of PSEUROT analyses suggest that the predominant conformation of the cyclopentane ring in 1−3 is defined by 128° < P < 140° for 1, 105° < P < 116° for 2, and 118° < P < 127° for 3, with the puckering amplitude in the range from 34° to 40° for 1−3. However, PSEUROT analyses based on our Karplus equation produced a smaller rms error by ≤0.14 Hz and ΔJmax error by ≤0.5 Hz than those performed with the standard Haasnoot−Altona equation. This work therefore highlights two important points: (i) the solution- and the solid-state structures of aristeromycin (1) are indeed different, and (ii) the close similarity of geometries derived from Haasnoot−Altona's equation or from our Karplus equation suggest that the solution structures for 1−3 are correctly defined.

Density functional calculations of the pseudorotational flexibility of tetrahydrofuran

The flexibility of the five-membered ring in tetrahydrofuran was investigated using quantum mechanical methods involving density functional, Hartree-Fock, and many-body perturbation theory (MP2, MP4) calculations. We found that motion along the pseudorotational path of tetrahydrofuran is nearly free. The 0.1 kcal/mol energy barrier for pseudorotation, calculated at the highest MP4(SDQ)/6-311++G(2d,p)//MP2/6-311++G(2d,p) level of theory, agrees well with the experimental value of 0.16±0.03 kcal/mol. Similar results were obtained with the S-VWN, B3-LYP and B-LYP density functional calculations using the 6-31G(d) set of atomic orbitals. Also the density functional dipole moments and geometries were in good agreement with both the MP2 and experimental benchmarks. However, all density functional methods that utilized the default integration grid in the Gaussian 94 program were found to provide false stationary points of the C 1 symmetry near the pseudorotational angle of 100°. These stationary points disappeared when a denser spherical-product grid was used. Overall, the hybrid B3-LYP functional was found to be the most promising quantum mechanical method for the modeling of biomolecules containing the furanose ring.

(2,3- O -Isopropylidene-5- O -(triphenylmethyl)-α-D-ribofuranosyl]-triphenylstannane

The synthesis, IR, MS, solution and solid state NMR spectra and X-ray structure of (2,3-O-isopropylidene-5-O-(triphenylmethyl)-α-D-ribofuranosyl]triphenylstannane 1 (R3Sn) are reported. Compound, 1 (R3Sn) crystallizes in the orthorhombic space group C2221 (Z = 8), with a = 8.935(4), b = 17.058(7), c = 47.94(5)A. There are two short tin–oxygen separations, at 3.030(7) [involving O4, on an alpha carbon] and 3.094(7) A [involving O2, on a beta carbon]. Only the O4 atom is considered to form a weak and additional bond to tin; this results in a deviation of the geometry at tin toward trigonal bypramid from tetrahedral, with O4 and C34 in the axial sites [C34– Sn–O4=152.3(3)°]. The conformation of the ribofuranosyl ring in the solid state is E o [P, the phase angle of pseudorotation, = 268.4(9)° and τ max , the puckering amplitude, = 38.0(6)°], while the isopropylidene ring conformation is between the envelope form, E C6, and the twist form, O3 T C6 [P = 46.5(10)° and τ max = 36.9(6)°]. In solution, the ribofuranosyl ring adopts a conformation between E o and 1 T o.

Nanosecond Molecular Dynamics Study of a Polycation Ribonucleic Guanidine (RNG) Duplexed with a Complementary DNA Oligomer Strand

Replacement of the phosphodiester linkages of the polyanion RNA with guanidinium linkers provides the polycation ribonucleic guanidine (RNG). Molecular dynamics studies to 1080 ps of the hybrid duplex formed from an octameric RNG strand r(Ag)8 with a complementary DNA oligomer strand d(Tp)8 have been carried out with explicit water solvent and Na+Cl- counterions under periodic boundary conditions with use of the CHARMM force field and Ewald summation method. The Watson−Crick hydrogen bonding pattern of the A/T tracts was maintained intact through the simulation without any structural restraints. Even for the two ends, only one end was partially frayed. The overall structure is equilibrated at a B-DNA conformation, and the RNG strand takes on the general conformation of the DNA backbone. The structural history of the RNG·DNA duplex was determined by examining histograms from the dynamics run. The histograms are of the hydrogen bonding pattern (sequence recognition) and base pair opening occurrences, minor groove width narrowing (3 Å), and bending of tracts. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 33° for the averaged structure. Propeller twist (associated with three-centered hydrogen bonding) up to −30°, native to DNA AT base pairing, was also observed. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3‘-endo domain. However, for the RNG strand the pseudorotation angles converted to O4‘-endo.

Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy

The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain.

Two Methylated Ribonucleosides: 3-Methyluridine and 1-Methylinosine

3-Methyluridine, C 10 H 14 N 2 O 6 , (1), and 1-methylinosine, C 11 H 14 N 4 O 5 , (2), adopt conformations generally consistent with those of published ribonucleoside and ribonucleotide crystal structures. (1) has a C(2')-endo ribofuranose pucker (Altona-Sundaralingam pseudorotation angle P = 175.5°); the glycosidic conformation is anti (X CN = -133.1°). (2) has two molecules in the asymmetric unit of which both are C(2')-endo (P = 159.8, 156.9°) with syn glycosidic conformations (X CN = 67.2, 53.6°).

Conformation of MgATP Bound to 5-phospho-alpha-d-ribose 1-diphosphate Synthetase by Two-dimensional Transferred Nuclear Overhauser Effect Spectroscopy

The conformation of MgATP bound at the active site of Salmonella typhimurium 5-phospho-α-d-ribose 1-diphosphate synthetase (PRibPP synthetase) has been investigated by two-dimensional transferred-NOE spectroscopy (TRNOESY). Inter-proton NOEs of the ligand were measured in the presence of the protein at several mixing times in the range of 40–300 ms at 500 MHz and 10°C. Measurements were made at low ligand concentrations (≈1 mM) in order to avoid weak non-specific ligand–protein interactions and to ensure that the NOE arises from the ligand bound at the active site. The inter-proton distances were determined from the experimentally observed NOE buildup curves by comparing them with theoretical simulations obtained by using the complete relaxation matrix. These distances were used as constraints in molecular modeling and energy minimization calculations to deduce the structure of the bound ligand. PRibPP synthetase is known to appreciably aggregate so that it exists in multiple oligomeric forms in solution. The structure was determined under the assumption that the ligand assumes the same conformation on each subunit of every oligomer regardless of its size. On the basis of the rotational correlation time deduced for the enzyme-nucleotide complexes, it is estimated that the average oligomer of PRibPP synthetase, in the sample used for the TRNOESY measurements, consists of about 30 subunits, whereas the smallest active form of the protein is a pentamer. The conformation of enzyme-bound MgATP is described by a glycosidic torsion angle χ= 50±5° and phase angle of pseudorotation P= 114.9° corresponding to a 1T° sugar pucker. It is noteworthy that the value of the glycosidic torsion angle obtained in this pyrophosphoryl transfer enzyme complex agrees well with those obtained previously for MgATP complexes of creatine kinase, pyruvate kinase (active and ancillary sites), and arginine kinase. The sugar pucker, on the other hand, differs from one enzyme complex to another.

Conformation of MgATP Bound to 5‐phospho‐α‐d‐ribose 1‐diphosphate Synthetase by Two‐dimensional Transferred Nuclear Overhauser Effect Spectroscopy

The conformation of MgATP bound at the active site of Salmonella typhimurium 5-phospho-alpha-D-ribose 1-diphosphate synthetase (PRibPP synthetase) has been investigated by two-dimensional transferred-NOE spectroscopy (TRNOESY). Inter-proton NOEs of the ligand were measured in the presence of the protein at several mixing times in the range of 40-300 ms at 500 MHz and 10 degrees C. Measurements were made at low ligand concentrations (approximately 1 mM) in order to avoid weak non-specific ligand-protein interactions and to ensure that the NOE arises from the ligand bound at the active site. The inter-proton distances were determined from the experimentally observed NOE buildup curves by comparing them with theoretical simulations obtained by using the complete relaxation matrix. These distances were used as constraints in molecular modeling and energy minimization calculations to deduce the structure of the bound ligand. PRibPP synthetase is known to appreciably aggregate so that it exists in multiple oligomeric forms in solution. The structure was determined under the assumption that the ligand assumes the same conformation on each subunit of every oligomer regardless of its size. On the basis of the rotational correlation time deduced for the enzyme-nucleotide complexes, it is estimated that the average oligomer of PRibPP synthetase, in the sample used for the TRNOESY measurements, consists of about 30 subunits, whereas the smallest active form of the protein is a pentamer. The conformation of enzyme-bound MgATP is described by a glycosidic torsion angle chi = 50 +/- 5 degrees and phase angle of pseudorotation P = 114.9 degrees corresponding to a 1T degree sugar pucker. It is noteworthy that the value of the glycosidic torsion angle obtained in this pyrophosphoryl transfer enzyme complex agrees well with those obtained previously for MgATP complexes of creatine kinase, pyruvate kinase (active and ancillary sites), and arginine kinase. The sugar pucker, on the other hand, differs from one enzyme complex to another.

Solution Structure of the Monoalkylated Mitomycin C–DNA Complex

Mitomycin C (MC) is a potent antitumor antibiotic which alkylates DNA through covalent linkage of its C-1″ position with the exocyclic N2amino group of guanine to yield the [MC]dG adduct at the duplex level. We report on the solution structure of the monoalkylated MC-DNA 9-mer complex where the [MC]dG5 adduct is positioned opposite dLC14 in the d(A3-C4-[MC]G5-T6).d(A13-C14-G15-T16) sequence context. The solution structure was solved based on a combined NMR-molecular dynamics study including NOE intensity based refinement. The formation of the [MC]dG adduct occurs with retention of the Watson-Crick alignment at the [MC]dG5.dC14 base-pair and flanking pairs in the complex. The MC ring is positioned in the minor groove with its indoloquinone aromatic ring system at a ∼45° angle relative to the helix axis and directed towards the 3′-direction on the unmodified strand. The MC indoloquinone chromphore is asymmetrically positioned in a slightly widened minor groove so that its plane is parallel to and stacked over the d(C14-G15-T16) segment on the unmodified strand with its other face exposed to solvent. The MC five-membered ring adopts an envelope pucker with its C-2″ atom displaced from the main plane and directed away from the unmodified strand. We observe conformational perturbations in the DNA 9-mer duplex on formation of the monoalkylated MC complex. Specifically, the base-pairs are displaced by ∼−3.0 Å towards the mjor groove on positioning the MC in the minor groove. This perturbation is accompanied by base stacking patterns similar to those observed inA-DNA while the majority of the sugars adopt puckers characteristic ofB-DNA. Conformational perturbations as monitored by helix twist, sugar pucker pseudorotation and glycosidic torsion angles are also observed for th 6(T6-C7-I8).d(C11-G12-A13) segment that is adjacent to but does not overlap the MC binding on the 9-mer duplex. We note that the O-10″ atom on the carbamate side-chain of MC forms an intermolecular hydrogen bond with the exocyclic amino group of dG15 in two of the three refined structures of the complex. The solution structure of the complex containing this intramolecular hydrogen bond readily explains both the previously observed d(C-G).d(C-G) sequence requirement for cross-linking and the observed, somewhat less stringent, requirement of the same sequence for the initial monoalkylation step. The structure indicates that the former is determined by the specific alignment of the MC monoadduct in the minor groove, while the specificity of the monoalkylation step likely reflects a specific hydrogen-bond between MC and DNA at the d(C-G).d(C-G) step in the precovalent complex.

Theoretical study on the deoxyribose radicals formed by hydrogen abstraction

The structures of C[sub 1], C[sub 2], C[sub 3], and C[sub 4] centered deoxyribose radicals are optimized at the UHF/6-31G level. For each radical two puckered conformations are observed, which are significantly different from those of deoxyribose. In C[sub 1] and C[sub 2] radicals the pseudorotation angle, P, is shifted toward higher values compared to that of deoxyribose. In C[sub 3] and C[sub 4] radicals the shift of P is in the opposite direction. In addition, the puckering amplitude [tau][sub m] is significantly decreased in the radicals, indicating a flattening of the five-membered ring. The C[sub 2] centered radical has the sp[sup 2]-type electronic structure, while C[sub 1], C[sub 3], and C[sub 4] centered radicals are pyramidal. The enthalpies for H-atom abstraction from deoxyribose are calculated at the MP2/6-31G[sup *] level including ZPE and a scaling of correlation energy. The abstractions from C[sub 1], C[sub 3], and C[sub 4] positions require similar energies, whereas the abstraction from the C[sub 2] carbon of deoxyribose is less favorable by 3-4 kcal/mol. The susceptibility of different deoxyribose hydrogens for abstraction is estimated on the basis of their bond strength and their accessibility in nucleotides and in DNA. 33 refs., 3 figs., 7 tabs.

Molecular Structure of Cyclic Diguanylic Acid at 1 Å Resolution of Two Crystal Forms: Self-association, Interactions with Metal Ion/Planar Dyes and Modeling Studies

Cyclic ribodiguanylic acid, c-(GpGp), is the endogenous effector regulator of cellulose synthase. Its three dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by x-ray diffraction analysis at 1 A resolution. Both structures were solved by direct methods and refined by block-matrix least squares refinement to R-factors of 0.112 (tetragonal) and 0.119 (trigonal). In both crystal forms, two independent c-(GpGp) molecules associate with each other to form a self-intercalated dimer. All four c-(GpGp) molecules have very similar backbone conformation. The riboses are in the C3'-endo pucker with pseudorotation angles ranging from -7.2 degrees to 16.5 degrees and the bases have anti glycosyl chi angles (-175.5 degrees to 179.7 degrees). In the tetragonal form, a hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (33 degrees). This metal coordination mechanism has been noted previously in other Pt- or Co-GMP complexes and may be relevant to the binding of the anticancer drug cisplatin to a GpG sequence in DNA. A model of the adduct between cisplatin and a d(CAATGGATTG) duplex has been constructed in which the induced bending of the DNA helix at the Pt crosslinking site is 33 degrees, consistent with earlier electrophoretic analyses. Moreover, c-(GpGp) exhibits unusual spectral properties not seen in other cyclic dinucleotides. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage-like model consisting of a tetrameric c-(GpGp) aggregate in which a large cavity (host molecule) is generated to afford a binding site for certain planar intercalators (guests molecules). The aggregate likely uses a hydrogen bonding scheme the same as that found in the G-quartet molecules, e.g., telomere DNA. The conformation of c-(GpGp) also suggests that certain nearest-neighbor intercalators may be synthesized on the basis of its unique molecular framework. Modeling studies have been carried out to test this hypothesis.

Structural effects of the C2-methylhypoxanthine:cytosine base pair in B-DNA: A combined NMR and X-ray diffraction study of d(CGC[m2I]AATTCGCG).

C2-Methylhypoxanthine (m2I) is a synthetic analog of guanine with the N2-amino group replaced by a methyl group. We have studied the structural consequence of the m2I incorporation in DNA by a combination of X-ray crystallographic, NMR, and enzymatic analyses. The crystal structure of d(CGC[m2I]AATTCGCG) has been solved and refined to an R factor of 20.7% at 2.25-A resolution. In the DNA duplex, the two independent m2I:C base pairs maintain the Watson-Crick scheme. While the C2-methyl group of m2I is in van der Waals contact with the O2 of the base-paired cytosine, it only causes the base pair to have slightly higher propeller twist and buckle angles. Its solution structure was analyzed by the NMR refinement procedure SPEDREF [Robinson, H., & Wang, A. H.-J. (1992) Biochemistry 31, 3524-3533] using 2D nuclear Overhauser effect data. Two starting models, a relaxed fiber model and an X-ray model, were subjected to the NOE-constrained refinement using 1518 NOE cross-peak integrals to arrive at the final models with (NOE) R factors of 13.8% and 14.3%, respectively. The RMSD between the two refined models (all atoms included) is 1.23 A, which presently seems to be near the limit of convergence of NOE-based refinement. The local structures of the two models are in better agreement as measured by the RMSD of the dinucleotide steps, falling in the range 0.54-0.98 A. Both refined solution structures confirm that the m2I dodecamer structure is of the B-DNA type with a narrow minor groove at the AT region, as observed in the crystal. However, significant differences exist between the crystal and solution structures in parameters such as pseudorotation angles, propeller twist angles, etc. The solution structure tends to have a more uniform backbone conformation, an observation consistent with that concluded from the laser Raman study of d(CGCAAATTTGCG) [Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., & Thomas, G. J., J. (1988) Biochemistry 27, 931-938]. Three related dodecamers, d(CGCGAATTCGCG), d(CGC[m2I]AATTCGCG), and d(CGC[e6G]AATTCGCG), were tested as substrates for the restriction endonuclease EcoRI. The m2I dodecamer was active, but the e6G dodecamer was not. Our results illustrate the complementarity in terms of the structural information provided by the two methods, X-ray diffraction and NMR.

Synthesis of 2′,3′- cis-fused pyrrolidino-β-D-nucleosides and their conformational analysis by 500 MHz 1H-NMR

The unique “off-template” stereoselectivity in the intramolecular free radical cyclization of 3-aza-5-hexenyl endocyclic radical has been demonstrated for the first time through the synthesis of 2′,3′- cis-fused pyrrolidino-β-D-nucleosides, 19a, 20a, 21a, 22a, 23a, 35a, 36a, 37a and 38a, which are not hithertofore available by any known procedures. The 2′-linked chiral carbon (C c) in 19a, 20a, 21a and 22a has shown 25:1 to 10:1 stereoselectivity depending upon the bulk of its substituent. The 3′-linked chiral carbon (C c) in 35a, 36a and 37a, on the other hand, has shown only 4:1 to 2:1 stereoselectivity. Finally, a full conformational analysis of 2′,3′-cis-fused pyrrolidino-β-D-nucleosides 19a, 20a, 21a, 22a, 23a, 35a, 36a, 37a and 38a is reported using 1H-NMR at 500 MHz. The solution geometry of the furanose and the pyrrolidine rings were studied on the basis of vicinal proton-proton coupling constants using the concept of pseudorotation. The furanose rings in 19a – 23a have a geometry biased toward a South-type conformation [70 – 81 % S, 126° < P S < 131° (C1′-exo-C2′-endo), 28° < Ψ m < 32°, rms error ±0.05]. In compounds 35a – 38a, the riboses have a conformation biased toward the North-type [∼90 % N, 20° < P N < 38° (C3′-endo) and 27°< Ψ m < 30°, rms error ±0.05]. In compounds 19a – 22a, the pyrrolidine geometry is biased toward the North-type conformation (C2′-exo), with a phase angle of pseudorotation Q N ≈ −28° and a puckering amplitude Ψ * m ≈ 40° (rms error ±0.5). In compounds 35a – 37a, the pyrrolidine has a geometry biased toward a South-type conformation (C2′-endo), with Q S ≈ 175° and Ψ * m ≈ 39° (rms error ±0.8). The furanose and the pyrrolidine rings share the C2′C3′ bond and have correlated conformations. The pyrrolidine adopts a North-type conformation in 19a – 22a when the ribose is in a South-type conformation. In contrast, the pyrrolidine ring adopts a South-type conformation when the ribose is in a North-type conformation (in 35a – 37a). Compounds 19a – 23a and 35a – 38a show a preference for the γ + conformation (∼70%). The glycosyl conformation for all pyrrolidine-fused systems was found to be anti.

Investigation of Solution Structure of d(GAATTTAAATTC)2 by1H NMR, Molecular Dynamics, Mechanics, Refinement by Back-Calculation of the NOESY Spectrum and Analysis of This Structure Using X-ray Data

1H NMR spectroscopy, restrained molecular mechanics and dynamics and refinements after back-calculation of the NOESY spectrum have been performed to study the structure of the d(GAATTTAAATTC)2 duplex and to determine whether it is bent or not. It is found that the duplex adopts a B-type conformation; all sugar conformations belong to the C2' endo region and purines have a larger pseudorotation angle as compared to pyrimidines. The cross-strand AH2(n)-AH1' (m + 1) distance (where (n) and (m) are complementary residues), crucial for an anomalous A/T tract structure, is large on the TA step and gradually decreases at the 3' and 5' ends of the TTTAAA tract and follows the rules proposed previously [Chuprina, V.P., Lipanov, A.A., Fedoroff, O.Yu, Kim, S.G., Kintanar, A., and Reid, B.R., Proc. Natl. Acad. Sci. U.S.A. 88, 9087 (1991)]. The changes in this distance correlate with those in the T1 value for AH2 protons which we measured for several oligonucleotide sequences. A total number of about 250 interproton distance constraints were determined from NOESY spectra and were used for structure determination by molecular mechanics, dynamics and refinement by back-calculations. It is shown that these data are not enough to determine whether the duplex is bent or not. The whole family of B-type conformations including bent and straight structures fit well with the available NMR data. In principle, additional non-NMR data could be used in order to reduce the number of the allowable structures. The refinement of the structures with additional different non-NMR constraints (used as a driving force) on P-P or H1'-H1' minor groove width distances in the TA region shows a very good correlation between these distances and the angle of bending of the dodecamer. The more the minor groove width increases in the TA region the more the duplex is bent at the major groove of this region. On the other hand, there is also a very good correlation between P-P, H1'-H1' and AH2-H1' cross-strand distances as follows from analysis of X-ray B-type structures. These two correlations, together with the increased AH2-H1' cross-strand NMR distance in the TA region of the dodecamer indicate that the duplex should be characterized by a wider minor groove in the TA region and be bent in the major groove in this region.