Biomedical subjects

G A Leonard

Publications and source records attributed to G A Leonard.

At least 37 records · Page 2Linked to original sources

PubMed1996

The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold.

BACKGROUND: [corrected] Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution. The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Amino Acid Sequence↗

PubMed1996

The crystal structure analysis of d(CGCGAASSCGCG)2, a synthetic DNA dodecamer duplex containing four 4'-thio-2'-deoxythymidine nucleotides.

The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG), where S = 4'-thio-2'-deoxythymidine, has converged at R=0.201 for 2605 reflections with F > 2sigma(F) in the resolution range 8.0-2.4 A for a model consisting of the dodecamer duplex and 66 water molecules. A comparison of its structure with that of the native dodecamer d(CGCGAATTCGCG) has revealed that the major differences between the two structures is a change in the conformation of the sugar-phosphate backbone in the regions at and adjacent to the positions of the modified nucleosides. Examination of the fine structural parameters for each of the structures reveals that the thiosugars adopt a C3'-exo conformation in d(CGCGAASSCGCG), rather than the approximate C1'-exo conformation found for the analogous sugars in the structure of d(CGCGAATTCGCG). The observed differences in structure between the two duplexes may help to explain the enhanced resistance to nuclease digestion of synthetic oligonucleotides containing 4'-thio-2'-deoxynucleotides.

Base Sequence↗

PubMed1996

MAD Phasing Strategies Explored with a Brominated Oligonucleotide Crystal at 1.65A Resolution.

The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.

Journal Article↗

PubMed1996

A randomised dose escalation study of subcutaneous interleukin 2 with and without levamisole in patients with metastatic renal cell carcinoma or malignant melanoma.

We have examined the efficacy, toxicity and host immunological response of two different dose schedules of interleukin 2 (IL-2) given subcutaneously, daily for 3 months in patients with renal cell carcinoma (RCC) or metastatic melanoma (MM). We also examined the effect of adding the immune modulator levamisole to the two different schedules of IL-2. Thirty-nine patients were entered into two sequential phase I/II studies. Eighteen patients entered study 1 and were randomised to receive IL-2, 3 x 10(6) IU m-2 day-1, subcutaneously for 3 months with or without levamisole 50 mg t.d.s. p.o. on days 1-3 on alternate weeks. Twenty-one patients entered study 2 and were randomised to receive 5.4 x 10(6) IU m-2 day-1 subcutaneously for 3 months with or without levamisole 50 mg t.d.s. p.o. on days 1-3 on alternate weeks. Blood was taken for peripheral blood lymphocyte (PBL) phenotype analysis, and measurement of IL-2, soluble IL-2 receptor (sIL-2R) and neopterin concentration. Two patients with metastatic melanoma, one in each study, responded (11.8%); both received IL-2 alone. Observations of immunological parameters showed that treatment with subcutaneous IL-2 resulted in a significant rise in the percentage of PBLs bearing CD25, CD3/HLA-DR, CD56 and levels of IL-2 receptor and neopterin. The total white blood cell count (WBC) and total lymphocyte count rose significantly on day 18 compared with pretreatment levels. The addition of levamisole to either IL-2 schedule resulted in no significant changes in any immunological parameters. This study illustrates that prolonged subcutaneous IL-2 can be given safely in the outpatient setting. There was no evidence that levamisole acts as an immunomodulator in this study.

Adjuvants, Immunologic↗

PubMed1995

p-(4,6-Diamino-s-triazin-2-yl)aminophenylarsonic acid dihydrate.

The structure of the title compound C9 H11 AsN6O3-2H2O, contains a planar organic moiety bonded to pentavalent arsenic. The lattice is stabilized by a combination of aromatic stacking interactions and by a hydrogen-bonding network involving water molecules of crystallization.

Arsenicals↗

PubMed1995

Additional crystal forms of the E. coli class II fructose-1,6-bisphosphate aldolase.

We have obtained two additional crystal forms of the metal-dependent class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of elongated plates have unit-cell dimensions a = 73.4, b = 120.0, c = 190.1 A, orthorhombic space group P2(1)2(1)2(1). Monoclinic prisms have unit-cell dimensions a = 67.7, b = 104.3, c = 52.8 A, beta = 105 degrees, space group P2(1). Diffraction to slightly better than 3.0 A, has been observed for both forms using in-house and synchrotron facilities. These crystal forms may aid the structure solution of this enzyme by presenting additional forms for heavy-atom derivatization. These forms have multiple copies of the enzyme in the asymmetric unit and averaging methods might also be useful in the analysis.

Journal Article↗

PubMed1995

Self-association of a DNA loop creates a quadruplex: crystal structure of d(GCATGCT) at 1.8 A resolution.

BACKGROUND: The flexibility of DNA enables it to adopt three interconvertible types of duplex termed the A-, B- and Z-forms. It can also produce hairpin loops, triplex structures and guanine-rich quadruplex structures. Conformational flexibility assists in the tight packaging of DNA, for example in chromosomes. This is important given the large quantity of genetic information that must be packaged efficiently. Moreover, the ability of DNA to specifically self-associate or interact with complementary sequences is fundamental to many biological processes. Structural studies provide information about DNA conformation and DNA-DNA interactions and suggest features that might be relevant to how the molecule performs its biological role. RESULTS: We have characterized the structure of a synthetic heptanucleotide that folds into a novel loop structure. The loop is stabilized by association with a cation, by intra-strand hydrogen bonds between guanine and cytosine that are distinct from the normal Watson-Crick hydrogen bonds, and by van der Waals interactions. Two loops associate through the formation of four G.C pairs that exhibit pronounced base-stacking interactions. The formation of a symmetric A.A base pair further stabilizes loop dimerization. Stacking of the A.A pair on a symmetry-related A.A pairing assists the formation of a four-stranded assembly. A T.T pairing is also observed between symmetry-related loops. CONCLUSIONS: This analysis provides a rare example of an experimentally determined non-duplex DNA structure. It provides conformational detail relevant to the tight packaging or folding of a DNA strand and illustrates how a cation might modulate phosphate-phosphate repulsion in a tightly packed structure. The observation of base quartets involving G.C base pairs suggests a further structure to be considered in DNA-DNA interactions. The structure also provides detailed geometries for A.A and T.T base pairs.

Base Composition↗

PubMed1995

Do C-H...O hydrogen bonds contribute to the stability of nucleic acid base pairs?

The possible formation of inter-base C-H.O hydrogen bonds in A.T, A.U and certain non-Watson-Crick base pairs is examined. A geometrical analysis in conjunction with implications for the thermodynamic stability of the base pairs suggests that C-H.O hydrogen bonds could form in nucleic acid base pairs. They may alleviate destabilizing interactions that would arise if an unsatisfied hydrogen-bond acceptor were present and mediate secondary hydrogen-bonding effects in these base pairs.

Journal Article↗

PubMed1994

Crystal structure of a DNA duplex containing 8-hydroxydeoxyguanine-adenine base pairs.

The crystal structure of the oligonucleotide d(CGCAAATTO8GGCG), containing the chemically modified base 8-hydroxydeoxyguanine (O8G), has been determined at 2.5-A resolution and refined to a crystallographic R-factor of 16.8%. The B-type DNA helix contains standard Watson-Crick base pairs except at the mismatch sites, where O8G adopts a syn conformation and forms hydrogen bonds to adenine in the anti conformation. The thermodynamic stability of the duplex was found by UV melting techniques to be intermediate between the native oligonucleotide d(CGCAAATTTGCG) and an oligonucleotide containing A.G mispairs d(CGCAAATTGGCG). Comparison of the structure of the O8G(syn).A(anti) base pair with those of Watson-Crick base pairs has given a reason why O8G.A base pairs are not well repaired by DNA proofreading enzymes.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1994

Crystal and molecular structure of r(CGCGAAUUAGCG): an RNA duplex containing two G(anti).A(anti) base pairs.

BACKGROUND: Non-Watson-Crick base pair associations contribute significantly to the stabilization of RNA tertiary structure. The conformation adopted by such pairs appears to be a function of both the sequence and the secondary structure of the RNA molecule. G.A mispairs adopt G(anti).A(anti) configurations in some circumstances, such as the ends of helical regions of rRNAs, but in other circumstances probably adopt an unusual configuration in which the inter-base hydrogen bonds involve functional groups from other bases. We investigated the structure of G.A pairs in a synthetic RNA dodecamer, r(CGCGAAUUAGCG), which forms a duplex containing two such mismatches. RESULTS: The structure of the RNA duplex was determined by single crystal X-ray diffraction techniques to a resolution in the range 7.0-1.8A, and found to be an A-type helical structure with 10 Watson-Crick pairs and two G.A mispairs. The mispairs adopt the G(anti).A(anti) conformation, held together by two obvious hydrogen bonds. Unlike analogous base pairs seen in a DNA duplex, they do not exhibit a high propeller twist and may therefore be further stabilized by weak, reverse, three-center hydrogen bonds. CONCLUSIONS: G(anti).A(anti) mispairs are held together by two hydrogen of guanine and the N6 and N1 of adenine. If the mispairs do not exhibit high propeller twist they may be further stabilized by inter-base reverse three-centre hydrogen bonds. These interactions, and other hydrogen bonds seen in our study, may be important in modelling the structure of RNA molecules and their interactions with other molecules.

Animals↗

PubMed1994

Guanine-1,N6-ethenoadenine base pairs in the crystal structure of d(CGCGAATT(epsilon dA)GCG).

A single-crystal X-ray analysis of the synthetic oligomer d(CGCGAATT(epsilon dA)GCG) (epsilon dA = 1,N6-ethenoadenosine) has been carried out. The B-form duplex crystallizes in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 24.31 A, b = 39.65 A, and c = 63.05 A. Refinement has converged with R = 0.182 for 2837 reflections in the resolution range 7.0-2.25 A for a model consisting of the duplex, one Mg2+ ion, and 127 water molecules. The structure contains two G.epsilon dA base pairings which adopt a G(anti).epsilon dA(syn) conformation. The geometry of the two mispairs suggests that the G.epsilon dA pairing are held together by three interbase hydrogen bonds. These are N2(G)-H...N1(epsilon dA), N1(G)...N9(epsilon dA), and O6(G)...H-C8(epsilon dA). The last interaction serves to alleviate the destabilizing effect that would occur due to the presence of an unfulfilled hydrogen bond acceptor. A superposition of the G(4).epsilon dA-(21) base pair found in this structure and the Watson-Crick G(4).C(21) base pair observed in the native dodecamer d(CGCGAATTCGCG) indicates a significant difference in the sugar/phosphate backbone. However, the overall conformations of the two duplexes remain similar, suggesting that the modified base pairs are accommodated into the double helix mainly by alterations of the backbone conformation. Such structural rearrangement of the backbone, upon incorporation of epsilon dA, may provide a signal to the 3-methyladenine-DNA glycosylase that repairs such lesions.

Adenine↗

PubMed1993

The crystal structure of N4-methylcytosine.guanosine base-pairs in the synthetic hexanucleotide d(CGCGm4CG).

The structure of d(CGCGm4CG) were m4C = N4-methylcytosine has been determined by crystallographic methods. The crystals are multifaced prisms, with orthorhombic space group P2(1)2(1)2(1) and unit cell dimensions of a = 17.98, b = 30.77 and c = 44.75A. The asymmetric unit consists of one duplex of hexanucleotide and 49 waters. The R-factor is 0.189 for 1495 reflections with F > or = sigma(F) to a resolution limit of 1.8A. The double helix has a Z-DNA type structure which appears to be intermediate in structure to the two previously characterised structure types for Z-DNA hexamers. The two m4C.G base-pairs adopt structures that are very similar to those of the equivalent base-pairs in the structure of the native sequence d(CGCGCG) except for the presence of the methyl groups which are trans to the N3 atoms of their parent nucleotides and protrude into the solvent region. The introduction of the modified base-pairs into the d(CGCGCG) duplex appears to have a minimal effect on the overall base-pair morphology of the Z-DNA duplex.

Base Composition↗

PubMed1993

Crystal and molecular structure of d(CGTAGATCTACG) at 2.25 A resolution.

Single crystal X-ray diffraction methods have been used to determine the structure of the synthetic DNA dodecamer d(CGTAGATCTACG). The dodecamer presents a new crystal form of B-DNA in the monoclinic system, space group C2 with a = 64.83 A, b = 35.36 A, c = 25.35 A and beta = 92.24 degrees. Structure solution was by molecular replacement and the refinement used a combination of rigid body treatment, molecular dynamics simulated annealing and restrained least-squares methods. The refinement has been concluded with an R-factor of 13.8% for 2120 reflections (78% of what is theoretically available) with F > or = 2 sigma (F) in the resolution range 7.0 to 2.25 A. The asymmetric unit comprises a B-form duplex (24 nucleotides, molecular weight 7.2 kDa), 136.5 water molecules and a single magnesium ion at 50% occupancy. The DNA model has root-mean-square derivations from standard bond lengths of 0.011 A. The central part of the structure is d(GATC), a tetrad sequence involved in methylation and mismatch repair. A comparison with other structures containing such a tetrad indicates polymorphism at the tetrad level. In our structure a narrow minor groove with a pronounced hydration pattern is observed. Such features may be of importance in the recognition of the sequence by specific enzymes. A noteworthy feature of the hydration is the identification of several pentagonal arrangements of hydrogen bonding groups. Details of conformational parameters, base stacking patterns, intermolecular interactions and hydration are presented and comparisons with related structures are given.

Base Sequence↗

PubMed1993

Anthracycline-DNA interactions at unfavourable base-pair triplet-binding sites: structures of d(CGGCCG)/daunomycin and d(TGGCCA)/adriamycin complexes.

The structures of two hexanucleotide-anthracycline complexes d(CGGCCG)/daunomycin and d(TGGCCA)/adriamycin have been determined using single-crystal X-ray diffraction techniques. In both cases the anthracycline molecule is bound to non-preferred d(YGG) base-pair triplet sites. For both complexes the crystals are tetragonal and belong to the space group P4(1)2(1)2. Unit-cell dimensions are a = 28.07 (2), c = 53.35 (1) and a = 28.01 (1), c = 52.99 (1) A, respectively, and the asymmetric unit of both structures consists of one strand of DNA, one drug molecule and approximately 50 water molecules. For the d(CGGCCG) complex the refinement converged with an R factor of 0.21 for 1108 reflections with F >/= 2sigma(F) in the resolution range 7.0-1.9 A. For the complex involving d(TGGCCA) the final R value was 0.22 for 1475 reflections in the range 7.0-1.7 A with the same criterion for observed data. Both complexes are essentially isomorphous with related structures but differ in terms of the number of favourable van der Waals interactions of the amino sugars of the drug molecules with the DNA duplexes and the formation in the minor groove of heterodromic pentagonal arrangements of hydrogen bonds involving water molecules which link the amino sugars to the DNA. These differences in structure are used to rationalize the lack of affinity of daunomycin-type anthracyclines for d(YGG) and d(YGC) sites.

Journal Article↗

PubMed1992

The conformational variability of an adenosine.inosine base-pair in a synthetic DNA dodecamer.

A crystal structure analysis of the synthetic deoxydodecamer d(CGCAAATTIGCG) which contains two adenosine.inosine (A.I) mispairs has revealed that, in this sequence, the A.I base-pairs adopt a A(anti).I(syn) configuration. The refinement converged at R = 0.158 for 2004 reflections with F greater than or equal to 2 sigma(F) in the range 7.0-2.5A for a model consisting of the DNA duplex and 71 water molecules. A notable feature of the structure is the presence of an almost complete spine of hydration spanning the minor groove of the whole of the (AAATTI)2 core region of the duplex. pH-dependent ultraviolet melting studies have suggested that the base-pair observed in the crystal structure is, in fact, a protonated AH+ (anti).I(syn) species and that the A.I base-pairs in the sequence studied display the same conformational variability as A.G mispairs in the sequence d(CGCAAATTGGCG). The AH+(anti).I(syn) base-pair predominates below pH 6.5 and an A(anti).I(anti) mispair is the major species present between pH 6.5 and 8.0. The protonated base-pairs are held together by two hydrogen bonds one between N6(A) and O6(I) and the other between N1(A) and N7(I). This second hydrogen bond is a direct result of the protonation of the N1 of adenosine. The ultraviolet melting studies indicate that the A(anti).I(anti) base-pair is more stable than the A(anti).G(anti) base-pair but that the AH+(anti).I(syn) base pair is less stable than its AH+(anti).G(syn) analogue. Possible reasons for this observation are discussed.

Adenosine↗

PubMed1992

Conformation of guanine-8-oxoadenine base pairs in the crystal structure of d(CGCGAATT(O8A)GCG).

The structure of the synthetic deoxydodecamer d(CGCGAATT(O8A)GCG)2 (O8A = 8-oxoadenine) has been determined by single-crystal X-ray diffraction techniques. The oligonucleotide crystallizes in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 25.48 A, b = 41.84 A, and c = 64.91 A. The refinement has converged with an R-factor of 0.151 for 1119 reflections in the resolution range 8.0-2.25 A. Sixty-seven solvent molecules were located during the course of the refinement. The B-DNA helix consists of ten Watson-Crick base pairs and two guanine-8-oxoadenine (G.O8A) base pairs. In order to achieve hydrogen-bonding complementarity between the two bases, an unusual G(anti).O8A-(syn) wobble conformation is adopted. It is proposed that the G.O8A mispairs are held together by a network of four interbase hydrogen bonds which are the result of the formation of two reverse three-center hydrogen-bonding systems. These involve one carbonyl oxygen lone pair interacting with two hydrogen atoms. In a departure from previous observations of the characteristics of purine-purine anti-syn base pairs, lambda 1 and lambda 2, the angles between the glycosidic bonds and the C1'-C1' vector, are symmetric. A reassessment of the other purine-purine mispairs suggests that similar three-center hydrogen bonds may occur and make a contribution to stabilizing other base pairings.

Adenine↗

PubMed1992

Anthracycline binding to DNA. High-resolution structure of d(TGTACA) complexed with 4'-epiadriamycin.

Crystallographic methods have been applied to determine the high-resolution structure of the complex formed between the self-complementary oligonucleotide d(TGTACA) and the anthracycline antibiotic 4'-epiadriamycin. The complex crystallises in the tetragonal system, space group P4(1)2(1)2 with a = 2.802 nm and c = 5.293 nm, and an asymmetric unit consisting of a single DNA strand, one drug molecule and 34 solvent molecules. The refinement converged with an R factor of 0.17 for the 2381 reflections with F greater than or equal to 3 sigma F in the resolution range 0.70-0.14 nm. Two asymmetric units associate such that a distorted B-DNA-type hexanucleotide duplex is formed incorporating two drug molecules that are intercalated at the TpG steps. The amino sugar of 4'-epiadriamycin binds in the minor groove of the duplex and displays different interactions from those observed in previously determined structures. Interactions between the hydrophilic groups of the amino sugar and the oligonucleotide are all mediated by solvent molecules. Ultraviolet melting measurements and comparison with other anthracycline-DNA complexes suggest that these indirect interactions have a powerful stabilising effect on the complex.

Base Sequence↗

PubMed1990

Structural and thermodynamic studies on the adenine.guanine mismatch in B-DNA.

The structure of the synthetic dodecamer d(CGCAAATTGGCG) has been shown by single crystal X-ray diffraction methods to be that of a B-DNA helix containing two A(anti).G(syn) base pairs. The refinement, based on data to a resolution of 2.25 A shows that the mismatch base pairs are held together by two hydrogen bonds. The syn-conformation of the guanine base of the mismatch is stabilised by hydrogen bonding to a network of solvent molecules in both the major and minor grooves. A pH-dependent ultraviolet melting study indicates that the duplex is stabilised by protonation, suggesting that the bases of the A.G mispair are present in their most common tautomeric forms and that the N(1)-atom of adenine is protonated. The structure refinement shows that there is some disorder in the sugar-phosphate backbone.

Adenine↗