Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors

Abstract

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp¹⁰⁸Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.

Fig. 1. Mechanism of base editing by ABEs.

(A) A schematic representation of base editing by ABEs. The ABEs studied as a part of the current work consist of a Cas9n fused to an evolved TadA* protein. The binding of Cas9n to the target genomic locus unwinds the DNA double helix and exposes a small region of ssDNA. TadA* acts on this ssDNA and deaminates adenine (A) to form inosine (I), which is subsequently converted to guanine (G) through DNA repair and replication. (B) Overall chemical reaction catalyzed by ABEs.

Fig. 2. Structural changes in the TadA* mutants revealed through MD simulations.

(A) The A:T to G⋮C base editing efficiency of the monomeric and dimeric ABE7.10 at six different target As in HEK293T cells. Values and error bars reflect the mean and SD of three independent biological replicates performed on different days. (B) Residue level flexibility of TadA* shown in terms of the root mean squared fluctuation (RMSF) of the Cα atoms of the peptide backbone. The β4-β5 loop region is highlighted in blue, and each mutation is indicated with its respective location in the protein. (C) Representative clusters from the trajectory of TadA*0.1and TadA*2.1 superimposed on each other, with clusters color coded as indicated in (D). (E) Comparison of the secondary structure of zinc-dependent deaminases: TadA* and APOBECs. Helices and arrows denote the α helices and β strands, respectively. The β4-β5 loop of interest in this study that interacts with the polynucleotide substrate is highlighted in both cases.

Fig. 3. Analyses of the TadA*-DNA contacts.

Asteroid plots for (A) TadA*0.1-ssDNA, (B) TadA*1.1-ssDNA, (C) TadA*1.2-ssDNA, and (D) TadA*2.1-ssDNA complexes. Details of the conformational change of residue 108 when it is mutated from Asp (TadA*0.1) (E) to Asn (TadA*1.1and later) (F).

Fig. 4. Asteroid plot analysis of second-generation mutations.

(A) The first and second interaction shell around the three nucleotides in the active site of the TadA*2.1-ssDNA complex. The size of the node corresponds to the time in which the amino acid resides in the first/second shell. First round mutations are red, and second round mutations are orange. (B) Structural overlay of average structure of TadA*0.1-ssDNA and TadA*2.1-ssDNA complexes. This α5 helix has been highlighted to depict its overall movement toward the active site upon Asp¹⁴⁷Tyr mutation.

Fig. 5. ABE mutations lead to an increase in TadA* binding to the ssDNA.

(A) List of early generation mutations in TadA that were analyzed in this study. (B) The model of the TadA*-ssDNA complex simulated to determine the binding energy profile of the TadA* mutants. The binding-unbinding event was monitored using the collective variable (ξ) defined as the distance between the center of mass of the protein and DNA. (C) The free-energy profile of binding of the ssDNA to various TadA*s. For each TadA*-ssDNA complex, the average PMF is shown as a function of the continuously changing ξ values. The shaded regions around individual curves depict the standard deviation for four independent replicates of the umbrella sampling simulations. The error bars associated with the mean PMFs indicate the error calculated using the block-averaging method.

Fig. 6. Significance of Asn¹⁰⁸ for base editing.

(A) ABE constructs created by reverting the Asp¹⁰⁸Asn mutation in the higher generation ABEs. (B) RMSF of the Cα atoms of the TadA*1.2(N108D) and TadA*2.1(N108D) enzymes. (C) The free-energy profile of binding of the hybrid TadA*s to ssDNA. The shaded regions around individual curves depict the SD for four independent replicates of the umbrella sampling calculations. The error bars associated with the mean PMFs indicate the error calculated using block-averaging method. (D and E) A:T to G⋮C base editing efficiencies in HEK293T cells by the various ABEs at six different target As. Fold-decrease values upon reversion analysis of the Asp¹⁰⁸Asn mutation are indicated above the bars. Values and error bars reflect the mean and SD of three independent biological replicates performed on different days.