Determinants of Oligonucleotide Selectivity of APOBEC3B

Abstract

APOBEC3B (A3B) is a prominent source of mutation in many cancers. To date, it has been difficult to capture the native protein-DNA interactions that confer A3B's substrate specificity by crystallography due to the highly dynamic nature of wild-type A3B active site. We use computational tools to restore a recent crystal structure of a DNA-bound A3B C-terminal domain mutant construct to its wild type sequence, and run molecular dynamics simulations to study its substrate recognition mechanisms. Analysis of these simulations reveal dynamics of the native A3Bctd-oligonucleotide interactions, including the experimentally inaccessible loop 1-oligonucleotide interactions. A second series of simulations in which the target cytosine nucleotide was computationally mutated from a deoxyribose to a ribose show a change in sugar ring pucker, leading to a rearrangement of the binding site and revealing a potential intermediate in the binding pathway. Finally, apo simulations of A3B, starting from the DNA-bound open state, experience a rapid and consistent closure of the binding site, reaching conformations incompatible with substrate binding. This study reveals a more realistic and dynamic view of the wild type A3B binding site and provides novel insights for structure-guided design efforts for A3B.

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

“…Even though 2-pyrimidone and iodide ion themselves are not potent inhibitors, they interact with the amino acid residues found in wild-type A3B. We therefore envision that novel small molecules that achieve higher affinity to stabilize these non-productive conformations might serve as chemical inhibitors for A3B, especially given the remarkable conformational plasticity of the A3B active site as demonstrated by MD simulations here, as well as in our prior studies 19,36 . In this regard, the A3B-GL7 structures presented may contribute to the future development of potent A3B inhibitors, which we hypothesize will help to control tumor evolution and other mutational processes relevant to human diseases.…”

“…The system went through multi-step energy minimization, followed by gradual heating and multi-step equilibration with decreasing restraints. The detailed MD protocol used for this system is exactly the same as outlined in our recent publication 36 . The final production MD trajectories with three independent copies, 500 ns each, were generated in an NPT ensemble at 310 K without any restraints.…”

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

“…Even though 2‐pyrimidone and iodide ion themselves are not potent inhibitors (Figures S1 and S2), they interact with the amino acid residues found in wild‐type A3B. We therefore envision that novel small molecules that achieve higher affinity to stabilize these non‐productive conformations might serve as chemical inhibitors for A3B, especially given the remarkable conformational plasticity of the A3B active site as demonstrated by MD simulations here, as well as in our prior studies . In this regard, the A3B‐GL7 structures presented may contribute to the future development of potent A3B inhibitors, which we hypothesize will help to control tumor evolution and other mutational processes relevant to human diseases.…”

“…The system went through multi‐step energy minimization, followed by gradual heating and multi‐step equilibration with decreasing restraints. The detailed MD protocol used for this system is exactly the same as outlined in our recent publication . The final production MD trajectories with three independent copies, 500 ns each, were generated in an NPT ensemble at 310 K without any restraints.…”

MagnEdit—interacting factors that recruit DNA-editing enzymes to single base targets

“…This study describes a fundamentally different approach to single base editing through the use of non-covalent interactions to "attract" a DNA cytosine deaminase to a single target cytosine. A3B is desirable for this application because it is normally nuclear (not shuttling or cytoplasmic like related family members) (29,30,31,34,35) and, because of active site structural constraints (33,36,37), unlikely to elicit RNA level off-target editing events as documented recently for BE3 and A3A CBEs (3,4). A variety of techniques may be used in the future to identify additional APOBEC-interacting "baits" for the Mag-nEdit system (proteomic, genetic, and directed-evolution,).…”