Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization

Abstract

Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair (BER) pathway by cleaving the N-glycosidic bond that connects nucleobases to the sugar-phosphate backbone in DNA. AAG targets a range of structurally diverse purine lesions using nonspecific DNA-protein π-π interactions. Nevertheless, the enzyme discriminates against the natural purines and is inhibited by pyrimidine lesions. This study uses molecular dynamics simulations and seven different neutral or charged substrates, inhibitors, or canonical purines to probe how the bound nucleotide affects the conformation of the AAG active site, and the role of active site residues in dictating substrate selectivity. The neutral substrates form a common DNA-protein hydrogen bond, which results in a consistent active site conformation that maximizes π-π interactions between the aromatic residues and the nucleobase required for catalysis. Nevertheless, subtle differences in DNA-enzyme contacts for different neutral substrates explain observed differential catalytic efficiencies. In contrast, the exocyclic amino groups of the natural purines clash with active site residues, which leads to catalytically incompetent DNA-enzyme complexes due to significant reorganization of active site water. Specifically, water resides between the A nucleobase and the active site aromatic amino acids required for catalysis, while a shift in the position of the general base (E125) repositions (potentially nucleophilic) water away from G. Despite sharing common amino groups, the methyl substituents in cationic purine lesions (3MeA and 7MeG) exhibit repulsion with active site residues, which repositions the damaged bases in the active site in a manner that promotes their excision. Overall, we provide a structural explanation for the diverse yet discriminatory substrate selectivity of AAG and rationalize key kinetic data available for the enzyme. Specifically, our results highlight the complex interplay of many different DNA-protein interactions used by AAG to facilitate BER, as well as the crucial role of the general base and water (nucleophile) positioning. The insights gained from our work will aid the understanding of the function of other enzymes that use flexible active sites to exhibit diverse substrate specificity.

Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker

“…That said, with so many ribonucleotides incorporated into the genome that are susceptible to oxidative damage, it is likely there are also damaged rNMPs within the genome. For example, ribonucleotide abasic sites could be generated by either spontaneous hydrolysis of the RNA N-glycosidic bond or by glycosylase cleavage of oxidized rNMPs [11] , [63] . While the RNA glycosidic bond is stronger and, thus, less susceptible to spontaneous hydrolysis than DNA, even a small fraction of incorporated ribonucleotides becoming abasic could pose a threat to genomic stability if not repaired by APE1 [6] .…”

Heterogeneous Dynamical Environment at the Interface of a Protein–DNA Complex

“…They demonstrated that configurationally similar water-mediated contacts play a distinct role during the recognition process of the DNA by the transcription factors belonging to the ETS-Family. In an important recent work, Lenz and Wetmore showed the significance of the reorganizations of DNA bases and water around the active sites during the inhibition process of enzymes. Structural arrangements and energetics of the interfacial water around the Bam HI protein–DNA complex has been explored in detail using the grand canonical Monte Carlo (GCMC) method .…”

Computational studies of DNA repair: Insights into the function of monofunctional DNA glycosylases in the base excision repair pathway

“…Specifically, a combination of Monte Carlo simulations (OPLS‐AA), free energy perturbation and DFT calculations (B3LYP/6‐311++G(d,p)) revealed that AAG significantly prefers binding to Hx over canonical A, providing the first insight into the lack of canonical purine repair. Subsequently, MD (AMBER) simulations have been used to investigate differences in the AAG active site configuration upon binding seven different nucleotides, including neutral or charged substrates, inhibitors and the undamaged purines . These calculations revealed that neutral substrates share a common active site hydrogen bond with His136, which aids alignment of active site π–π interactions that are likely critical for catalysis.…”