A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs.

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb-A111N can form bidentate hydrogen bonds with the N3 and O4 moieties of the uracil base. Genetic analysis indicates the gain of function for A/U base pairs allows the MUG-K68N mutant to remove uracil incorporated into the genome during DNA replication. The implications of this study in the origin of life are discussed.

Specificity and catalytic mechanism in family 5 uracil DNA glycosylase.

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily.

Identification of Escherichia coli mismatch-specific uracil DNA glycosylase as a robust xanthine DNA glycosylase.

The gene for the mismatch-specific uracil DNA glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the human thymine DNA glycosylase with activity against mismatched uracil base pairs. Examination of cell extracts led us to detect a previously unknown xanthine DNA glycosylase (XDG) activity in E. coli. DNA glycosylase assays with purified enzymes indicated the novel XDG activity is attributable to MUG. Here, we report a biochemical characterization of xanthine DNA glycosylase activity in MUG. The wild type MUG possesses more robust activity against xanthine than uracil and is active against all xanthine-containing DNA (C/X, T/X, G/X, A/X and single-stranded X). Analysis of potentials of mean force indicates that the double-stranded xanthine base pairs have a relatively narrow energetic difference in base flipping, whereas the tendency for uracil base flipping follows the order of C/U > G/U > T/U > A/U. Site-directed mutagenesis performed on conserved motifs revealed that Asn-140 and Ser-23 are important determinants for XDG activity in E. coli MUG. Molecular modeling and molecular dynamics simulations reveal distinct hydrogen-bonding patterns in the active site of E. coli MUG that account for the specificity differences between E. coli MUG and human thymine DNA glycosylase as well as that between the wild type MUG and the Asn-140 and Ser-23 mutants. This study underscores the role of the favorable binding interactions in modulating the specificity of DNA glycosylases.

Analyzing the robustness of the MM/PBSA free energy calculation method: application to DNA conformational transitions.

The ability to predict and characterize free energy differences associated with conformational equilibria or the binding of biomolecules is vital to understanding the molecular basis of many important biological functions. As biological studies focus on larger molecular complexes and properties of the genome, proteome, and interactome, the development and characterization of efficient methods for calculating free energy becomes increasingly essential. The aim of this study is to examine the robustness of the end-point free energy method termed the molecular mechanics Poisson-Boltzmann solvent accessible surface area (MM/PBSA) method. Specifically, applications of MM/PBSA to the conformational equilibria of nucleic acid (NA) systems are explored. This is achieved by comparing A to B form DNA conformational free energy differences calculated using MM/PBSA with corresponding free energy differences determined with a more rigorous and time-consuming umbrella sampling algorithm. In addition, the robustness of NA MM/PBSA calculations is also evaluated in terms of the sensitivity towards the choice of force field and the choice of solvent model used during conformational sampling. MM/PBSA calculations of the free energy difference between A-form and B-form DNA are shown to be in very close agreement with the PMF result determined using an umbrella sampling approach. Further, it is found that the MM/PBSA conformational free energy differences were also in agreement using either the CHARMM or AMBER force field. The influence of ionic strength on conformational stability was particularly insensitive to the choice of force field. Finally, it is also shown that the use of a generalized Born implicit solvent during conformational sampling results in free energy estimates that deviate slightly from those obtained using explicitly solvated MD simulations in these NA systems.

Structure and dynamics of a fusion peptide helical hairpin on the membrane surface: comparison of molecular simulations and NMR.

The conserved N-terminal residues of the HA2 subunit of influenza hemagglutinin (fusion peptide) are essential for membrane fusion and viral entry. Recent NMR studies showed that the 23-residue fusion peptide forms a helical hairpin that undergoes rocking motion relative to the membrane surface on a nanosecond time scale. To compare with NMR and to obtain a detailed molecular picture of the peptide-membrane interaction, we performed molecular dynamics simulations of the fusion peptide in explicit dimyristoylphosphatidylcholine and with the IMM1 implicit membrane model. To account for low and neutral pH conditions, simulations were performed with acidic groups (E11 and D19) protonated and unprotonated, respectively. The hairpin structure was stable in the simulations, with the N-terminal helix buried more deeply into the hydrophobic membrane interior than the C-terminal helix. Interactions between the tryptophans in the fusion peptide and phospholipid residues contribute to peptide orientation. Higher flexibility of the hairpin was observed in the implicit membrane simulations. Internal correlation functions of backbone N-H vectors were fit to the extended Lipari-Szabo model-free approach to obtain order parameters and correlation times. Good agreement with the NMR results was obtained for orientational fluctuations around the hairpin axis (rotation), but those around the perpendicular axis (tilting) were more limited in the simulations than inferred from the NMR experiments.