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: MD simulations of the hotspot (AGCT) (Supplementary Video S1) and coldspot (GCCT) substrate complexes with AID-WT revealed differences in specific protein-DNA contacts. For this modeling, the underlying hypothesis is that perturbed interactions between a specific protein residue and DNA nucleotides result in reduced deaminase activity. Within this analytical framework, the distribution of residue-to-DNA time-averaged distances revealed that WT residues Tyr114 and Arg119 make consistent contacts with the hotspot substrate (Figure 6A). Conversely, only Tyr114 was found to consistently contact the coldspot substrate. With the hotspot substrate, Tyr114 formed aromatic stacking interactions with -1 Guanine throughout the trajectory, and occasionally wedged between the -1 Gua and -2 Ade. Arg119 formed significant hydrogen bonding interactions with -1 Gua N7/O6 and more transient electrostatic interactions with the phosphate linkage between -1 Gua and -2 Ade (Supplementary Table S1). The side chains of residues Leu113 and Phe115 are buried (Supplementary Table S2) and form hydrophobic contacts with one another that shape the surrounding protein architecture, positioning Tyr114 for stacking interactions and the backbone amide of Leu113 for potential hydrogen bonding interactions with the DNA. It should be noted that our unconstrained in silico tetranucleotide substrate displayed greater dynamics than might be expected with longer physiological substrates that would be constrained by both the upstream and downstream DNA. Although this presented the challenge of potentially destabilizing some intermolecular interactions, it also conferred the advantage of allowing for greater exploration of conformations and binding poses.
Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.