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

Kiran S. Gajula, Peter J. Huwe, Charlie Y. Mo, Daniel J. Crawford, James Stivers, Ravi Radhakrishnan, Rahul M. Kohli

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)9964-9975
Number of pages12
JournalNucleic Acids Research
Volume42
Issue number15
DOIs
StatePublished - Sep 1 2014

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Mutagenesis
DNA
Nucleotide Motifs
Proteins
Enzyme Activation
Antibodies
Cytosine
Molecular Dynamics Simulation
AICDA (activation-induced cytidine deaminase)
APOBEC Deaminases

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Gajula, K. S., Huwe, P. J., Mo, C. Y., Crawford, D. J., Stivers, J., Radhakrishnan, R., & Kohli, R. M. (2014). High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase. Nucleic Acids Research, 42(15), 9964-9975. https://doi.org/10.1093/nar/gku689

High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase. / Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James; Radhakrishnan, Ravi; Kohli, Rahul M.

In: Nucleic Acids Research, Vol. 42, No. 15, 01.09.2014, p. 9964-9975.

Research output: Contribution to journalArticle

Gajula, Kiran S. ; Huwe, Peter J. ; Mo, Charlie Y. ; Crawford, Daniel J. ; Stivers, James ; Radhakrishnan, Ravi ; Kohli, Rahul M. / High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase. In: Nucleic Acids Research. 2014 ; Vol. 42, No. 15. pp. 9964-9975.
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