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

Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM - Nucleic Acids Res. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

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Characterization of selected AID variants. (A) Amino acids which evolved to be greater than 20% of the total G3 library at each position were characterized for their activity using the rifampin mutagenesis assay and the in vitro deamination assay. The data are represented as in Figure 1. Experimental data that are significantly different from the AID-WT (P < 0.05) are highlighted with an asterisk (*). At the right of the plot are shown the results for cvBEST, which was selected based on covariation of the best amino acids at each position as described below. (B) To examine the impact of covariation of multiple positions in the loop, a starting pooled library was generated containing the WT amino acid at each position along with any other amino acid that resulted in >20% of the counts in G3 at that position. The covariation library underwent six cycles of selection and at the conclusion of selection, individual colonies were sequenced and the frequency of each amino acid variant was cataloged. The results are summarized as a normalized logo plot on bottom panel.
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Figure 4: Characterization of selected AID variants. (A) Amino acids which evolved to be greater than 20% of the total G3 library at each position were characterized for their activity using the rifampin mutagenesis assay and the in vitro deamination assay. The data are represented as in Figure 1. Experimental data that are significantly different from the AID-WT (P < 0.05) are highlighted with an asterisk (*). At the right of the plot are shown the results for cvBEST, which was selected based on covariation of the best amino acids at each position as described below. (B) To examine the impact of covariation of multiple positions in the loop, a starting pooled library was generated containing the WT amino acid at each position along with any other amino acid that resulted in >20% of the counts in G3 at that position. The covariation library underwent six cycles of selection and at the conclusion of selection, individual colonies were sequenced and the frequency of each amino acid variant was cataloged. The results are summarized as a normalized logo plot on bottom panel.

Mentions: We next aimed to understand why certain mutations were preferred in the Sat-Sel-Seq procedure. Across all positions, we selected mutants that represented >20% of the total count in G3 and evaluated these mutants in the context of our two complementary deaminase assays. In the rifampin assay, the majority of selected variants had activity equivalent to or greater than AID-WT, within the limits of statistical significance (Figure 4A). When the individual point mutants were purified and evaluated using the in vitro deamination assay, all of the variants that were favored over AID-WT in Sat-Sel-Seq showed increased deaminase activity. Among the variants, one notable mutation, R119G, demonstrated the largest enhancement in activity in both the rifampin assay (∼4-fold) and the in vitro enzymatic assay (at least 3-fold).


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

Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM - Nucleic Acids Res. (2014)

Characterization of selected AID variants. (A) Amino acids which evolved to be greater than 20% of the total G3 library at each position were characterized for their activity using the rifampin mutagenesis assay and the in vitro deamination assay. The data are represented as in Figure 1. Experimental data that are significantly different from the AID-WT (P < 0.05) are highlighted with an asterisk (*). At the right of the plot are shown the results for cvBEST, which was selected based on covariation of the best amino acids at each position as described below. (B) To examine the impact of covariation of multiple positions in the loop, a starting pooled library was generated containing the WT amino acid at each position along with any other amino acid that resulted in >20% of the counts in G3 at that position. The covariation library underwent six cycles of selection and at the conclusion of selection, individual colonies were sequenced and the frequency of each amino acid variant was cataloged. The results are summarized as a normalized logo plot on bottom panel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150791&req=5

Figure 4: Characterization of selected AID variants. (A) Amino acids which evolved to be greater than 20% of the total G3 library at each position were characterized for their activity using the rifampin mutagenesis assay and the in vitro deamination assay. The data are represented as in Figure 1. Experimental data that are significantly different from the AID-WT (P < 0.05) are highlighted with an asterisk (*). At the right of the plot are shown the results for cvBEST, which was selected based on covariation of the best amino acids at each position as described below. (B) To examine the impact of covariation of multiple positions in the loop, a starting pooled library was generated containing the WT amino acid at each position along with any other amino acid that resulted in >20% of the counts in G3 at that position. The covariation library underwent six cycles of selection and at the conclusion of selection, individual colonies were sequenced and the frequency of each amino acid variant was cataloged. The results are summarized as a normalized logo plot on bottom panel.
Mentions: We next aimed to understand why certain mutations were preferred in the Sat-Sel-Seq procedure. Across all positions, we selected mutants that represented >20% of the total count in G3 and evaluated these mutants in the context of our two complementary deaminase assays. In the rifampin assay, the majority of selected variants had activity equivalent to or greater than AID-WT, within the limits of statistical significance (Figure 4A). When the individual point mutants were purified and evaluated using the in vitro deamination assay, all of the variants that were favored over AID-WT in Sat-Sel-Seq showed increased deaminase activity. Among the variants, one notable mutation, R119G, demonstrated the largest enhancement in activity in both the rifampin assay (∼4-fold) and the in vitro enzymatic assay (at least 3-fold).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Show MeSH