Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.     

Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase beta. Correlation with DNA binding and dRP lyase activity

Mammalian DNA polymerase beta functions in the base excision DNA repair pathway filling in short patches (1-5 nt) in damaged DNA and removing deoxyribose 5'-phosphate from the 5'-side of damaged DNA. The backbone dynamics and the refined solution structure of the N-terminal domain of beta-Pol have been characterized in order to establish the potential contribution(s) of backbone motion to the DNA binding and deoxyribose 5'-phosphate lyase function of this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60 degrees crossing between the pairs. The RMSD of the NMR conformers (residues 13-80) is 0.37 A for the backbone heavy atoms and 0.78 A for all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12mer shows a consensus surface for the binding of these various DNA oligomers, that surrounds and includes the deoxyribose 5'-phosphate lyase active site region. Connection segments between helices 1 and 2 and between helices 3 and 4 each contribute to DNA binding. Helix-3-turn-helix-4 forms a helix-hairpin-helix motif. The highly conserved hairpin sequence (LPGVG) displays a significant degree of picosecond time-scale motion within the backbone, that is possibly important for DNA binding at the phosphodiester backbone. An Omega-loop connecting helices 1 and 2 and helix-2 itself display significant exchange contributions (R(ex)) at the backbone amides due to apparent conformational type motion on a millisecond time-scale. This motion is likely important in allowing the Omega-loop and helix-2 to shift toward, and productively interact with, gapped DNA. The deoxyribose 5'-phosphate lyase catalytic residues that include K72 which forms the Schiff's base, Y39 which is postulated to promote proton transfer to the aldehyde, and K35 which assists in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assists in the opening of the hemiacetal, shows conformational exchange.     

Side-chain dynamics of the SAP SH2 domain correlate with a binding hot spot and a region with conformational plasticity

X-linked lymphoproliferative disease is caused by mutations in the protein SAP, which consists almost entirely of a single SH2 domain. SAP interacts with the Tyr281 site of the T<-->B cell signaling protein SLAM via its SH2 domain. Interestingly, binding is not dependent on phosphorylation but does involve interactions with residues N-terminal to the Tyr. We have used 15N and 2H NMR relaxation experiments to investigate the motional properties of the SAP SH2 domain backbone amides and side-chain methyl groups in the free protein and complexes with phosphorylated and non-phosphorylated peptides derived from the Tyr281 site of SLAM. The most mobile methyl groups are in side-chains with large RMSD values between the three crystal structures of SAP, suggesting that fast time-scale dynamics in side-chains is associated with conformational plasticity. The backbone amides of two residues which interact with the C-terminal part of the peptides experience fast time-scale motions in the free SH2 domain that are quenched upon binding of either the phosphorylated or non-phosphorylated peptide. Of most importance, the mobility of methyl groups in and around the binding site for residues in the N-terminus of the peptide is significantly restricted in the complexes, underscoring the dominance of this interaction with SAP and demonstrating a correlation between changes in rapid side-chain motion upon binding with local binding energy.     

The DNA-binding domain in the Bacillus subtilis transition-state regulator AbrB employs significant motion for promiscuous DNA recognition

AbrB is a Bacillus subtilis protein responsible for regulating a diverse array of unrelated genes during periods of sub-optimal growth conditions. DNA binding by AbrB is unique in that sequence recognition is specific, yet no obvious consensus sequence of bound promoter regions is apparent. The N-terminal domain is a recently characterized representative of a novel class of DNA-binding proteins that possess a looped-hinge helix DNA-binding topology. Although the structural characterization of this DNA-binding topology contributed to an understanding of the architectural basis for recognition of DNA target sequences, specific mechanisms responsible for promiscuity in DNA sequence recognition still were not apparent. Analysis of (15)N backbone relaxation parameters shows that dynamic motion of regions directly linked to DNA binding show concerted motion on the microsecond-millisecond timescale. Furthermore, dynamic motion of the hinge region suggests that the DNA-binding region is capable of conformational orientations that allow it to accommodate DNA sequence variability in the cognate binding sites.     

Structural characterization of the Fpg family of DNA glycosylases

Until recently, the Fpg family was the only major group of DNA glycosylases for which no structural data existed. Prototypical members of this family, found in eukaryotes as well as prokaryotes, have now been crystallized as free proteins and as complexes with DNA. In this review, we analyze the available structural information for formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei). Special emphasis is placed on mechanisms by which these enzymes recognize and selectively excise cognate lesions from oxidatively damaged DNA. The problem of lesion recognition is considered in two parts: how the enzyme efficiently locates a single lesion embedded in a vast excess of DNA; and how the lesion is accommodated in a pocket near the active site of the enzyme. Although all crystal structures reported to date for the Fpg family lack the damaged base, functionally important residues that participate in DNA binding and enzyme catalysis have been clearly identified and other residues, responsible for substrate specificity, have been inferred.     

Changes in structure and dynamics of the Fv fragment of a catalytic antibody upon binding of inhibitor

Binding of the product inhibitor p-nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable-domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant changes in the chemical shifts of the backbone resonances upon binding extend beyond the immediate vicinity of the antigen binding site into the interface between the two associated polypeptides that form the Fv heterodimer, a possible indication that the binding of ligand causes a change in the relative orientations of the component light (V(L)) and heavy (V(H)) chain polypeptides. Significant differences in backbone dynamics were observed between the free Fv and the complex with p-nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen-binding site showed less broadening of resonances, but still required exchange terms (R(ex)) in the model-free dynamics analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p-nitrophenol caused these resonances to sharpen, but some R(ex) terms are still required in the analysis of the backbone dynamics. We conclude that the slow timescale motions in the antigen-binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large-amplitude motions by the bound inhibitor.     

Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization

Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop beta3-beta4 and loopbeta4-beta5, and include Ile18. The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop beta3-beta4 also markedly contribute to binding. Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the "RNA chaperone" activity of CspB.     

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.     

Distinct repair activities of human 7,8-dihydro-8-oxoguanine DNA glycosylase and formamidopyrimidine DNA glycosylase for formamidopyrimidine and 7,8-dihydro-8-oxoguanine

7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells. Both lesions are repaired by human 7, 8-dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively. In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The k(cat)/K(m) values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg. However, the k(cat)/K(m) values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. Analysis of the Schiff base intermediate by NaBH(4) trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases. Surprisingly, such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair. The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg. The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K(m)), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.     

Pre-steady-state kinetic study of substrate specificity of Escherichia coli formamidopyrimidine--DNA glycosylase

Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied stopped-flow kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG.C), poor substrates (8-oxoG.A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG.C, the complex enters the productive catalytic reaction after approximately 50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition.     

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.     

Probing Slow Backbone Dynamics in Proteins Using TROSY-Based Experiments to Detect Cross-Correlated Time-Modulation of Isotropic Chemical Shifts

The difference in the relaxation rates of zero-quantum (ZQ) and double-quantum (DQ) coherences is the result of three principal mechanisms. These include the cross-correlation between the chemical shift anisotropies of the two participating nuclei, dipolar interactions with remote protons as well as interference effects due to the time-modulation of their isotropic chemical shifts as a consequence of slow micros-ms dynamics. The last effect when present, dominates the others resulting in large differences between the relaxation rates of ZQ and DQ coherences. We present here four sets of TROSY-based (Salzmann et al., 1998) experiments that measure this effect for several pairs of backbone nuclei including (15)N, (13)C(alpha) and (13)C'. These experiments allow the detection of the presence of slow dynamic processes in the protein backbone including correlated motion over two and three bonds. Further, we define a new parameter chi which represents the extent of correlated motion on the slow (micros-ms) timescale. This methodology has been applied to (15)N,(13)C,REDPRO-(2)H-labeled (Shekhtman et al., 2002) human ubiquitin. The ubiquitin backbone is seen to exhibit extensive dynamics on the slow timescale. This is most pronounced in several residues in the N-terminal region of the alpha-helix and in the loop connecting the strands beta(4) and beta(5). These residues which include Glu24, Asn25, Glu51 and Asp 52 form a continuous surface. As an additional benefit, the measured rates confirm the dependence of the (13)C(alpha) chemical shift tensor on local secondary structure of the protein backbone.     

Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups.

Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.     

Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylase

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity.     

Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility

Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei-DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 A resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 A) and Nei-R252A (2.05 A). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a approximately 50 degrees rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme.     

Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamics

Formamidopyrimidine-DNA glycosylase (Fpg) identifies and removes 8-oxoguanine from DNA. All of the X-ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C-terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X-ray structure of Lactococcus lactis Fpg bound to an abasic site analog-containing DNA and of Bacillus stearothermophilus Fpg bound to 8-oxoG. This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8-oxoG in its binding site. In addition, from our results, the conformation of the 8-oxoG seems to be favored in syn in the L. lactis models, in agreement with the available X-ray structure from B. stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region.     

Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

Formamidopyrimidine-DNA-glycosylase (Fpg pro tein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2′-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation.