Insights into conformational changes in AlkD bound to DNA with a yatakemycin adduct from computational simulations

Abstract

Structural integrity of DNA molecules is necessary for their information storage function. Cells rely on a number of pathways to ensure that the damage to DNA induced by endogenous and exogenous reagents is repaired. AlkD, a base excision enzyme, removes a damaged nucleobase by cleaving a glycosidic bond. Unlike many other base excision enzymes, AlkD does not flip a damaged nucleobase into a designated reaction pocket, and as such can repair nucleobases with larger adducts, such as yatakemycin. In this study, the structure and dynamics of AlkD have been investigated by classical molecular dynamics simulations. Several systems including apo-AlkD, and AlkD in complex with DNA, both with and without the yatakemycin adduct have been simulated. Comparison of the results for the apo-AlkD with AlkD with substrate (damaged or undamaged) indicates a high degree of motion of helix αB in apo-AlkD, whereas this helix is observed to form various contacts when the substrate is bound. The calculated results are consistent with previous experimental studies that have suggested various residues involved in damage recognition, DNA binding, and base excision catalysis.

Characterization of the Search Complex and Recognition Mechanism of the AlkD–DNA Glycosylase

“…The AlkD–DNA PC1 then highlights the W187–A*6 contact, which has also been demonstrated to be a main component of the AlkD repair machinery by Parsons et al Next, the Y27–A7 hydrogen bond distance between sidechain and the ring nitrogen, N3, represents one of two residues that extends from the opposite side of the AlkD-binding cleft to interact with the nucleobase 3′ to the methylation site. Although Y27 does not insert directly between base pairs of the DNA, it is a partially intercalating residue, which is generally an integral part of canonical glycosylase repair. , Moreover, Y27 is thought to play a role in the screening process, and thus this result is consistent with this hypothesis . Another DNA backbone hydrogen-bonding interaction is then seen with R26–T9, which extends along the DNA from the same α-helix as Y27.…”

“…12 Moreover, the scanning tyrosine of OGG1 discovered using fluorescent kinetic analysis is comparable to the αβ helix Y27, which has been proposed to be relevant to the search process. 8,28 Indeed, the Y27−A7 interaction is demonstrated here to be crucial for separating the AlkD SC from the EC. And although the exact role of Y27 in scanning nucleotides is not known, it is demonstrated that in the SC the Y27−A7 hydrogen bond is from Y27 to the O4′ ribose oxygen of A7 (Figures S11 and S12).…”

“…Kinetics experiments in conjunction with fluorescent detection reveal the importance of selected hydrophobic residues in MutM and OGG1, and likewise AlkD is also found to have crucial hydrophobic residues participating in the recognition event. , The combination of W109, W187, and F180 is analogous to these glycosylases, which have hydrophobic residues in their active site, and in the case of MutM, use these residues to probe DNA in the IC . Moreover, the scanning tyrosine of OGG1 discovered using fluorescent kinetic analysis is comparable to the αβ helix Y27, which has been proposed to be relevant to the search process. , Indeed, the Y27–A7 interaction is demonstrated here to be crucial for separating the AlkD SC from the EC. And although the exact role of Y27 in scanning nucleotides is not known, it is demonstrated that in the SC the Y27–A7 hydrogen bond is from Y27 to the O4′ ribose oxygen of A7 (Figures S11 and S12).…”

“…3,50 Moreover, Y27 is thought to play a role in the screening process, and thus this result is consistent with this hypothesis. 28 Another DNA backbone hydrogen-bonding interaction is then seen with R26−T9, which extends along the DNA from the same α-helix as Y27. Finally, the F180 C−H/π interaction with A7 proves important in distinguishing the EC and SC.…”

“…It has been suggested, due to the high fluctuation of the αβ helix in apo-AlkD simulation and the potential of Y27 to interact with substrate, that this helix may play a significant role in the search process (Y27 can be seen in the inlet of Figure 1). 28 Additionally, the backbone root-meansquare deviation (RMSD) between the crystallized apo structure of AlkD and the catalytic 3mA-bound structure is less than 0.5 Å, indicating little to no structural change in the protein upon binding of a DNA lesion. Crystal structures of AlkD bound to various product states including an abasic site, 20 a dissociated 3mA, 21 and a product state of the bulky adenine lesion YTMA 23 also demonstrate little change in protein structure.…”