High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

Abstract

Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function.

DNA cytosine and methylcytosine deamination by APOBEC3B: enhancing methylcytosine deamination by engineering APOBEC3B

“…The first possibility is that the loop-1 sequence/conformation may influence the conformation of the highly conserved Tyr 313 on loop-7 of A3BCD2, which may determine how well the target mC residue can get to and bind at the active site. Previous reports suggest that Tyr 114 of AID and Tyr 315 of A3G, which are equivalent to Tyr 313 of A3B, may also participate in positioning the target C [ 48 , 67 ]. Therefore, Tyr 313 of A3B may bear dual functions: stabilize target C and discriminate mC at the active pocket.…”

In silico analysis of the V66M variant of human BDNF in psychiatric disorders: An approach to precision medicine

“…Mutator Plugin1 . 3 [50], available in the Visual Molecular Dynamics (VMD) 4.5 software package [51], was used to induce the V66M substitution on the validated model of BDNF.…”

In silico analysis of PFN1 related to amyotrophic lateral sclerosis

“…MD simulations of the wild-type PNF1 and its natural variants: C71G, M114T, E117G and G118V, were performed using the GROMACS 2018.2 package [40]. Mutator Plugin 1.3 [41], which is available in the Visual Molecular Dynamics (VMD) 1.9.1 software [42] was used to induce the C71G, M114T, E117G and G118V substitution on the experimentally determined structure of wild type PFN1 (PDB ID: 1PFL) [29].…”