High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.
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.
Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.Show MeSH
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Mentions: The rifampin mutagenesis assay and the in vitro deamination assay demonstrated similar activity patterns in alanine scanning mutagenesis (Figure 1). Within the loop, the N-terminal residues Leu113, Tyr114 and Phe115 appeared essential in both the assays, with activities comparable to the negative controls. The central residues spanning Cys116 to Lys120, along with Pro123, were generally tolerant of alanine mutations, though all showed decreased activity relative to AID-WT. Both the A121G and E122A mutants showed decreased activity relative to AID-WT, although curiously this manifests to a greater extent with the in vitro assay than with the rifampin-based bacterial assay. While the patterns are consistent, the differences in the assays points to the importance of using complementary assays to measure deaminase function. Differences could either be related to cellular factors altering protein activity in the rifampin assay or to altered in vitro properties of purified deaminases, such as aggregation. Taken together, the consensus of the two approaches suggested the essentiality of the N-terminal region, with more flexibility in the central and C-terminal regions of the protein loop.
Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.