title: 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.
authors: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors: Kiran S
authors: KS
authors: Gajula
authors: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors: Peter J
authors: PJ
authors: Huwe
authors: Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors: Charlie Y
authors: CY
authors: Mo
authors: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors: Daniel J
authors: DJ
authors: Crawford
authors: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors: James T
authors: JT
authors: Stivers
authors: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors: Ravi
authors: R
authors: Radhakrishnan
authors: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA rkohli@upenn.edu.
authors: Rahul M
authors: RM
authors: Kohli
mesh_terms: D000409:Alanine / Q000235:genetics; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D004247:DNA; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase; D000409:Alanine