Base-Displaced Intercalated Structure of the 3-(2-Deoxy-ß-D-erythropentofuranosyl)-pyrimido[1,2-f]purine-6,10(3H,5H)-dione (6-oxo-M1dG) Lesion in DNA.

The genotoxic 3-(2-deoxy-ß-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) DNA lesion arises from endogenous exposures to base propenals generated by oxidative damage and from exposures to malondialdehyde (MDA), produced by lipid peroxidation. Once formed, M1dG may oxidize, in vivo, to 3-(2-deoxy-ß-D-erythropentofuranosyl)-pyrimido[1,2-f]purine-6,10(3H,5H)-dione (6-oxo-M1dG). The latter blocks DNA replication and is a substrate for error-prone mutagenic bypass by the Y-family DNA polymerase hpol η. To examine structural consequences of 6-oxo-M1dG damage in DNA, we conducted NMR studies of 6-oxo-M1dG incorporated site-specifically into 5' -d(C1A2T3X4A5T6G7A8C9G10C11T12)-3':5'-d(A13G14C15G16T17C18A19T20C21A22T23G24)-3' (X = 6-oxo-M1dG). NMR spectra afforded detailed resonance assignments. Chemical shift analyses revealed that nucleobase C21, complementary to 6-oxo-M1dG, was deshielded compared with the unmodified duplex. Sequential NOEs between 6-oxo-M1dG and A5 were disrupted, as well as NOEs between T20 and C21 in the complementary strand. The structure of the 6-oxo-M1dG modified DNA duplex was refined by using molecular dynamics (rMD) calculations restrained by NOE data. It revealed that 6-oxo-M1dG intercalated into the duplex and remained in the anti-conformation about the glycosyl bond. The complementary cytosine C21 extruded into the major groove, accommodating the intercalated 6-oxo-M1dG. The 6-oxo-M1dG H7 and H8 protons faced toward the major groove, while the 6-oxo-M1dG imidazole proton H2 faced into the major groove. Structural perturbations to dsDNA were limited to the 6-oxo-M1dG damaged base pair and the flanking T3:A22 and A5:T20 base pairs. Both neighboring base pairs remained within the Watson-Crick hydrogen bonding contact. The 6-oxo-M1dG did not stack well with the 5'-neighboring base pair T3:A22 but showed improved stacking with the 3'-neighboring base pair A5:T20. Overall, the base-displaced intercalated structure was consistent with thermal destabilization of the 6-oxo-M1dG damaged DNA duplex; thermal melting temperature data showed a 15 °C decrease in Tm compared to the unmodified duplex. The structural consequences of 6-oxo-M1dG formation in DNA are evaluated in the context of the chemical biology of this lesion.

The peroxidation-derived DNA adduct, 6-oxo-M1dG, is a strong block to replication by human DNA polymerase η.

The DNA adduct 6-oxo-M1dG, (3-(2'-deoxy-ß-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the peroxidation-derived adduct M1dG. However, the effect of 6-oxo-M1dG adducts on subsequent DNA replication is unclear. Here we investigated the ability of the human Y-family polymerase hPol η to bypass 6-oxo-M1dG. Using steady-state kinetics and analysis of DNA extension products by liquid chromatography-tandem mass spectrometry, we found hPol η preferentially inserts a dAMP or dGMP nucleotide into primer-templates across from the 6-oxo-M1dG adduct, with dGMP being slightly preferred. We also show primer-templates with a 3'-terminal dGMP or dAMP across from 6-oxo-M1dG were extended to a greater degree than primers with a dCMP or dTMP across from the adduct. In addition, we explored the structural basis for bypass of 6-oxo-M1dG by hPol η using X-ray crystallography of both an insertion-stage and an extension-stage complex. In the insertion-stage complex, we observed that the incoming dCTP opposite 6-oxo-M1dG, although present during crystallization, was not present in the active site. We found the adduct does not interact with residues in the hPol η active site but rather forms stacking interactions with the base pair immediately 3' to the adduct. In the extension-stage complex, we observed the 3' hydroxyl group of the primer strand dGMP across from 6-oxo-M1dG is not positioned correctly to form a phosphodiester bond with the incoming dCTP. Taken together, these results indicate 6-oxo-M1dG forms a strong block to DNA replication by hPol η and provide a structural basis for its blocking ability.

Site-Specific Synthesis of Oligonucleotides Containing 6-Oxo-M1dG, the Genomic Metabolite of M1dG, and Liquid Chromatography-Tandem Mass Spectrometry Analysis of Its In Vitro Bypass by Human Polymerase ι.

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M1dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M1dG in genomic DNA is enzymatically oxidized to 6-oxo-M1dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M1dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M1dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M1dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M1dG blocks further replication by the enzyme.

Catalytic site amino acids of PKGI-alpha influence allosteric cGMP binding.

Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (K(a)) of each mutant protein for phosphotransferase activation and allosteric (3H)cGMP-binding affinity (K(D)) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (K(m)) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.

Structural determinants for calcium mobilization by prostaglandin E2 and prostaglandin F2alpha glyceryl esters in RAW 264.7 cells and H1819 cells.

2-Arachidonoylglycerol is oxygenated by cyclooxygenase-2 to form prostaglandin glyceryl esters. Previous work in this laboratory has suggested that PGE(2)-G activates a novel G protein-coupled receptor in a murine macrophage-like cell line, RAW 264.7. To probe the structural determinants for the putative receptor in RAW 264.7 cells, a panel of 10 analogs was tested for their ability to increase intracellular calcium. These analogs included PGE(2)- and PGF(2alpha)-ethanolamide, 4 PGE(2) glyceryl ester analogs, and 4 PGF(2alpha) glyceryl ester analogs. The glyceryl ester analogs differed in the positioning of the hydroxyl groups in the glycerol moiety and the type of linker (ester, amide, or thioester) of the prostaglandin to the glycerol moiety. Compounds were also evaluated in a human non-small cell lung cancer cell line (H1819). The glycerol moiety was required for the calcium response. All glyceryl ester analogs but not ethanolamides caused a concentration-dependent increase in calcium levels in both RAW 264.7 and H1819 cells. An amide or ester linkage was preferable to a thioester linkage. The EC(50) values did not significantly change when the positioning of the hydroxyls was varied. This calcium response induced by the glyceryl ester analogs appears to be independent of the putative hydrolysis products, PGE(2) and PGF(2alpha), and appears to represent a novel signaling pathway.

Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes.

Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGIalpha-(Delta349-670) and PKGIbeta-(Delta364-685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 A, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and K(D) values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGIalpha-isolated R domain and contraction of the PKGIbeta-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGIbeta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIalpha R domain may partially account for the elongation of wild type PKGIalpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.

Dimerization of cGMP-dependent protein kinase Ibeta is mediated by an extensive amino-terminal leucine zipper motif, and dimerization modulates enzyme function.

All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Ibeta this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats ((L3A/L10A/L17A/I24A), (L31A/I38A/L45A/I52A), (L17A/I24A/L31A/I38A/L45A/I52A), and (L3A/L10A/L45A/I52A)) of the motif produces a monomeric PKG Ibeta. Mutation of two Leu/Ile heptad repeats can produce either a dimeric (L3A/L10A) or monomeric (L17A/I24A and L31A/I38A) PKG. Point mutation of Leu-17 or Ile-24 (L17A or I24A) does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Ibeta. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions (Leu-17, Ile-24, Leu-31, and Ile-38) appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants (PKG Ibeta (delta1-52) and PKG Ibeta L17A/I24A/L31A/I38A/L45A/I52A) is 2- to 3-fold greater than that for wild type dimeric PKG Ibeta, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site (site A) of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme.

Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase.

Using small-angle x-ray scattering, we have observed the cGMP-induced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (Delta(1-52)PKG-I beta). On saturation with cGMP, the radius of gyration of Delta(1-52)PKG-I beta increases from 29.4 +/- 0.1 A to 40.1 +/- 0.7 A, and the maximum linear dimension increases from 90 A +/- 10% to 130 A +/- 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to Delta(1-52)PKG-I beta indicates that elongation of Delta(1-52)PKG-I beta requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of Delta(1-52)PKG-I beta. We also predict that there is little or no cooperativity in cGMP binding to the two sites of Delta(1-52)PKG-I beta under the conditions used here. Results obtained by using the Delta(1-52)PKG-I beta monomer indicate that a previously observed elongation of PKG-I alpha is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-I beta activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.

Mechanisms of autoinhibition in cyclic nucleotide-dependent protein kinases.

Cyclic AMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) are autoinhibited through multiple interactions between their respective regulatory and catalytic domains. A large portion of this autoinhibition occurs through interactions between residues within the catalytic domain and those within either a substrate-like sequence (-RRXSX-) or pseudosubstrate sequence (-RRXAX-) in the regulatory domains. These contacts effectively inhibit catalysis by blocking substrate binding. Particularly important contacts involve the P-2, P-3, and P+1 residues where either serine, which is potentially autophosphorylated, or alanine occupies the P0 position. The primary sequence is apparently less important for autoinhibition in PKGs than in PKAs, and a conserved serine at P+2 in PKGs is important for autoinhibitory contacts. Elements outside the substrate-related sequences also contribute to autoinhibition in both PKA and PKG. For example, synthetic peptides with relatively short pseudosubstrate sequences are weak inhibitors; while heat-denatured RII subunit does not inhibit catalytic subunit, it is still rapidly autophosphorylated; and truncated PKGs lacking the substrate-like sequence are still partially autoinhibited. Thus, capacity for autoinhibition of PKA or PKG is provided by contacts involving direct interactions with the catalytic site and by contacts that stabilize an inactive conformation.