Brian E. Hingerty

Topography of cyclodextrin inclusion complexes. XII. Hydrogen bonding in the crystal structure of α-cyclodextrin hexahydrate: the use of a multicounter detector in neutron diffraction

In the crystalline complex ct-cyclodextrin hexahydrate, C36H60Oa0.6H20, orthorhombic, space group P2~212 ~, Z = 4, a = 14.858 (3), b = 34.038 (7), c = 9.529 (2) A, thirty hydroxyl hydrogen atoms are present, including one which is disordered (occupancies 0 .92 :0 .08) and could not be located with certainty from X-ray counter data. 851 low-angle neutron data * For part XI, see Proceedings of the International Symposium on Biomolecular Structure, Conformation, Function and Evolution, Madras 1978, to be published. For part X, see Saenger, Noltemeyer, Manor, Hingerty & Klar (1976) and Cramer, Bergmann, Manor, Noltemeyer & Saenger (1976). ~ Present address: SCS Scientific Control Systems GmbH, Oehleckerring 40, D-2000 Hamburg 62, Federal Republic of Germany. 0567-7408/80/051154-12501.00 were measured using a conventional four-circle diffractometer with a graphite monochromator (2 = 2.384 A) and a neutron flux rate of 6.4 x 104 n mm -2 s -1. Further, 1957 high-angle neutron data were collected by means of the multicounter device hedgehog. This instrument contains 100 BF 3 counters mounted on rotatable and extendable arms on the inside of a sphere, at the centre of which is installed a conventional Eulerian cradle holding the sample crystal. With the application of a modified Laue method, integration is over wavelength rather than over crystal rocking motion; a wavelength band of 1.275 < 2 < 1.31 A, a Cu-crystal monochromator, and a neutron flux of 2.4 x 104 n mm -2 s -1 were used. Details of the measurement and of the data reduction are given, and the refinement process, including combined neutron and a new set of 4268 X-ray data, is described. R factors are

DNA Adducts from a Tumorigenic Metabolite of Benzo[a]pyrene Block Human RNA Polymerase II Elongation in a Sequence- and Stereochemistry-dependent Manner

Many carcinogens exert their cancer-causing effects by reacting with DNA either directly or following metabolic activation, resulting in covalently linked combination molecules known as carcinogen-DNA adducts. The presence of such lesions in the genome increases the error frequency of the replication machinery, causing mutations that contribute to the initiation and progression of cancer. Cellular DNA repair pathways remove carcinogen adducts from DNA, thus averting the mutagenic potential of many DNA lesions by reducing their presence in the genome. Bulky DNA adducts, like those derived from a number of activated environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), are primarily repaired by the nucleotide excision repair (NER) pathway. Transcription-coupled NER (TC-NER) preferentially removes lesions from the transcribed strand of actively expressed genes, and RNA polymerase II stalled at the lesion quite possibly initiates the pathway. Among the bulky DNA adducts that are subject to TC-NER are those resulting from the reaction of the metabolically activated PAH benzo[a]pyrene (BP) with DNA. The P450 mixed-function oxygenases convert BP into a number of reactive intermediates, including tumorigenic (+)- and non-tumorigenic (-)-anti-benzo[a]pyrene diol epoxide (BPDE) that react with DNA via trans epoxide opening to form (+)-trans-anti-[BP]-N(2)-dG ((+)-ta[BP]G) and (-)-trans-anti-[BP]-N(2)-dG ((-)-ta[BP]G), respectively. To test the effect of these lesions on RNA synthesis, in vitro transcription assays using human nuclear extracts were performed with DNA templates containing an RNAPII promoter and a stereochemically pure (+)- or (-)-ta[BP]G adduct on the transcribed or non-transcribed strand. Transcription past (+)- or (-)-ta[BP]G adducts was investigated in the same sequence context to examine stereochemical effects. The (+)-ta[BP]G adduct was investigated in two different local sequence contexts to determine if the surrounding bases influence the adducts ability to block transcription. These experiments revealed that (+)- and (-)-ta[BP]G adducts on the transcribed strand of the DNA template block RNAPII in a sequence and stereochemistry-dependent manner; however, adducts on the non-transcribed strand do not block elongation significantly but may increase pausing at innate pause sites. In order to elucidate biologically influential differences between the (+)- and (-)-ta[BP]G structures, the DUPLEX program was used to carry out potential energy minimization searches at model transcription junctions. The lowest-energy minimum for the (+)-ta[BP]G adduct gives a structure in which the benzo[a]pyrenyl ring system resides in the minor groove of the heteroduplex region. In contrast, the lowest-energy minimum for a (-)-ta[BP]G adduct shows an orientation in which the benzo[a]pyrenyl group adopts a carcinogen/base-stacked conformation. These conformational preferences may contribute to the differential treatment of (+)- and (-)-ta[BP]G adducts by human RNAPII. In addition, while previous experiments showed that BPDE adducts cause T7RNAP to produce a ladder of truncated transcripts, RNAPII is blocked entirely at only one or two positions by the (+)- and (-)-ta[BP]G adducts, depending on sequence context. It is likely that these differences between the behaviors of T7RNAP and human RNAPII are a result of the structural characteristics of the enzymes active sites, a hypothesis that is explored in light of their known crystal structures.

Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3′ end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

The crystal and molecular structure of a calcium salt of guanylyl-3',5'-cytidine (GpC)

The calcium salt, Ca(C19H24NaOI2P)2.18H20, of guanylyl-3,5-cytidine (GpC) has been refined to an R of 8.2 % for 2918 observed reflections (11% for 4237 reflections, including unobserved). The molecule crystallized in space group P2~ with a= 21 224, b = 34207, c= 9327/~,, fl= 90.527 °, Z= 4. The asymmetric unit contains four GpC, 36 waters and two C a 2+ ions, for a total of 198 non-hydrogen atoms. The four GpC occur as two dimers related by a pseudo C-face-centering. Each dimer consists of two crystallographically independent GpC as Watson-Crick base-pairs, and possesses a pseudo twofold axis broken by a Ca 2+ ion and associated solvent. The structure was solved by an unusual series of steps including semi-empirical potential-energy methods, packing analysis, rigid-body refinement, least-squares and difference Fourier techniques, and direct-methods tangent-formula phase refinement. The four GpC have conformational angles in the range of helical RNA, but are not identical. The different crystallographic environments perturb the GpC from exact symmetry and demonstrate the range of the basic helical conformations. All eight bases are anti, sugars are all C(Y) endo, the C(4)-C(5) bond rotations are gauche-gauche, and the co,co angle pair about the O-P bonds is gauche--gauche-.

Solution Structure of the Aminofluorene [AF]-External Conformer of the anti-[AF]-C8-dG Adduct Opposite dC in a DNA Duplex†

The Escherichia coli genome contains a C-G1-G2-C-G3-C-C NarI hot spot sequence for -2 deletion mutations at G3 by aromatic amine carcinogens 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) that form covalent adducts at the C8-position of the guanine ring. Each of the three guanines are positioned in different sequence contexts (C-G1-G, G-G2-C, and C-G3-C) which provides an opportunity to investigate the potential sequence dependent interconversion between AF-intercalated and AF-external conformers of the [AF]dG adduct positioned opposite dC within the NarI sequence at the duplex level. We have prepared and purified DNA duplexes containing the [AF]dG adduct positioned in C-[AF]G-G, G-[AF]G-C, and C-[AF]G-C NarI sequence contexts and observe the ratio of AF-intercalated to AF-external conformers to be 30:70, 10:90, and 50:50, respectively. We have applied a combined NMR-molecular mechanics approach to define the structure of the AF-external conformer in the G-[AF]G-C NarI sequence context where it is the predominant conformation (90%) in solution. The modified guanine of the [AF]dG adduct aligns through Watson-Crick pairing with its partner cytosine and is stacked into the helix between flanking Watson-Crick dG.dC base pairs. The AF-external conformer with its anti-[AF]dG residue causes minimal perturbations in the DNA duplex at and adjacent to the lesion site with the covalently linked fluorenyl ring readily accommodated in the major groove and tilted toward the 5-end of the modified strand of the helix. This paper on the structure of the AF-external conformer with an anti-[AF]dG adduct together with the preceding paper in this issue on the structure of the AF-intercalated conformer with a syn-[AF]dG adduct defines for the first time the capacity of the mutagenic [AF]dG lesion to adopt interconverting syn and antialignments with the equilibrium shifting between the conformers depending on nearest neighbor and next-nearest neighbor sequences. Perhaps, recognition of the [AF]dG lesion by the repair machinery would be able to discriminate between the AF-intercalated conformer with its base displacement-fluorenyl ring insertion perturbation of the helix and the AF-external conformer where the DNA helix is essentially unperturbed at the lesion site and the fluorenyl ring is positioned with directionality in the major groove.

Accurate representation of B-DNA double helical structure with implicit solvent and counterions.

High-resolution nuclear magnetic resonance (NMR) and crystallographic data have been taken to refine the force field used in the torsion angle space nucleic acids molecular mechanics program DUPLEX. The population balance deduced from NMR studies of two carcinogen-modified DNA conformers in equilibrium was used to fine tune a sigmoidal, distance-dependent dielectric function so that reasonable relative energies could be obtained. In addition, the base-pair and backbone geometry from high-resolution crystal structures of the Dickerson-Drew dodecamer was used to re-evaluate the deoxyribose pseudorotation profile and the Lennard-Jones nonbonded energy terms. With a modified dielectric function that assumes a very steep distance-dependent form, a deoxyribose pseudorotation profile with reduced energy barriers between C2- and C3-endo minima, and a shift of the Lennard-Jones potential energy minimum to a distance approximately 0.4 A greater than the sum of the van der Waals radii, the sequence-dependent conformational features of the Dickerson-Drew dodecamer in both the solid state and the aqueous liquid crystalline phase are well reproduced. The robust performance of the revised force field, in conjunction with its efficiency through implicit treatment of solvent and counterions, provides a valuable tool for elucidating conformations and structure-function relationships of DNA, including those of molecules modified by carcinogens and other ligands.

Minor-groove binding models for acetylaminofluorene modified DNA

Minimized potential energy calculations have been employed to locate and evaluate energetically a number of different models for DNA modified at carbon-8 of guanine by acetylaminofluorene (AAF). Three different duplex nonamer sequences were investigated. In addition to syn guanine models which have some denaturation and a Z-DNA model, we have found two new types of structures in which guanine remains syn and the AAF is placed in the minor groove of a B-DNA helix. One type features Hoogsteen base pairing between the modified guanine and protonated cytosine, with a sharply bent helix. The other (here termed the wedge model because the aromatic amine is wedged into the minor groove) maintains a single hydrogen bond between O6 of the modified guanine and N3 of protonated cytosine, with much less deformation of the helix, and close Van der Waals contacts between the AAF and the walls of the minor groove. Both types of structures (as well as the related forms produced by deprotonation of cytosine) are energetically important in all three sequences examined. The wedge-type model, which is most favored except in alternating G-C sequences, has been previously observed in a combined NMR and computational characterization of an aminofluorene (AF) modified guanine opposite adenine in a DNA duplex undecamer (D. Norman, P. Abuaf, B.E. Hingerty, D. Live, D. Grunberger, S. Broyde and D.J. Patel, Biochemistry 28, 7462 (1989)).

Energy minimized structures of carcinogen-DNA adducts: 2-acetylaminofluorene and 2-aminofluorene.

Energy minimized structures of DNA modified by the aromatic amines 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF), for which no experimental atomic resolution data exist, are presented. These have been computed with a new molecular mechanics program specifically designed to define distortions imposed by such adducts, and employing a rational strategy for searching the conformation space of a DNA molecule with covalently linked carcinogen. In alternating G-C sequences, the AAF adduct prefers to reside at the exterior of an undeformed Z-helix. It can also induce base displacement with attendant denaturation and helix bending in sequences that disfavor the Z form, but undeformed B helices are excluded. The AF adduct, by contrast, prefers the major groove of an unperturbed B-helix, but can also induce carcinogen-base stacking in single stranded regions of the DNA, such as at the replication fork. The different biological properties of these two adducts may be related to their distinct

Neutron Diffraction of Alpha, Beta and Gamma Cyclodextrins: Hydrogen Bonding Patterns

Cyclodextrins (CDs) have proved useful as model systems for the study of hydrogen bonding. They are torus-shaped molecules composed of six(alpha), seven(beta) or eight(gamma) (1----4) linked glucoses. Because of their particular geometry, they are able to act as a host to form inclusion complexes with guest molecules very much like enzymes. Cyclodextrins have been shown to exert catalytic activity on suitable included-substrate molecules; they catalyze the hydrolysis of phenylacetates, of organic pyrophosphates and of penicillin derivatives. They also accelerate aromatic chlorinations and diazo coupling by means of their primary and/or secondary hydroxyl groups, so that the rates of hydrolysis are enhanced by up to a factor of 400. In order to understand the hydrogen bonding in these enzyme models, neutron diffraction data were collected to unambiguously determine the hydrogen atom positions, which could not be done from the x-ray diffraction data. alpha-CD has been shown to have two different structures with well-defined hydrogen bonds, one tense and the other relaxed. An induced-fit-like mechanism for alpha-CD complex formation has been proposed. Circular hydrogen bond networks have also been found for alpha-CD due to the energetically favored cooperative effect. beta-CD with a disordered water structure possesses an unusual flip-flop hydrogen bonding system of the type O-H...H-O representing an equilibrium between two states: O-H...O in equilibrium O...H-O. gamma-CD with a disordered water structure similar to beta-CD also possesses the flip-flop hydrogen bond. This study demonstrates that hydrogen bonds are operative in disordered systems and display dynamics even in the solid state.

Helix geometry of single stranded DNA ‘A’ and ‘B’ forms from minimum energy conformations of dimeric subunits

Low energy conformations with dihedral angles similar to those occurring in fibers of the A and B forms of DNAs have been calculated for the deoxydinucleoside phosphates dApdA, dCpdC, dTpdT, dGpdG and dGpdC (1-3). These conformers have been used as building blocks for generating larger single stranded polymers, whose helical parameters we have calculated. We find that single stranded A and B form helices tend to be narrower and more tightly wound than the duplexes obtained in fibers (4,5). This is consistent with experimental observations on single stranded fibers of poly (rC) (6). We also find that the different sequences have different helix geometries. In addition, it is observed that large variations in helix geometry for a given sequence are achievable at little energetic cost.