Model-guided mutational analysis of Activation-induced cytidine deaminase (AID) to elucidate structure-function relationships

King, Justin (2020) Model-guided mutational analysis of Activation-induced cytidine deaminase (AID) to elucidate structure-function relationships. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Activation-induced cytidine deaminase (AID) is a DNA mutating cytidine deaminase enzyme that is a member of the AID/ Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases, altogether consisting of 11 members in humans. AID deaminates deoxycytidine to deoxyuridine at immunoglobulin loci in activated B lymphocytes to mediate secondary antibody diversification. AID-mediated mutations have also been implicated in epigenetic reprogramming, as well as genome-wide DNA damage and chromosomal translocations that transform healthy B cells into lymphoma/leukemia. Since its discovery in 1999, a key element missing in the AID field has been the lack of a protein structure. The goal of this thesis was to solve this problem and determine how AID binds its substrate nucleic acid and how these interactions result in its physiological and aberrant activities. In chapter 2 of this thesis, we explored the role AID plays in epigenetic reprogramming through genome-demethylation. We provided a glimpse into the AID catalytic pocket structure and how AID from humans and other species target cytidine vs 5-methylcytidine. In chapter 3, we provided the first functional and breathing structure of AID using a unique computational-biochemical approach. In this work, we provided the first fine map of AID’s structure, identified the structural details of its catalytic pocket and interactions with the DNA substrate including secondary catalytic residues that are key to cytidine stabilization in the pocket, delineated two novel substrate DNA binding grooves on the surface and studied how conformational changes in the AID structure regulate its function. The work culminated in our discovery and proof that dynamic catalytic pocket closure limits AID activity. This novel catalytic pocket state which we termed the “Schrödinger’s CATalytic pocket” has since been confirmed by multiple other structural studies and represents a novel mode of enzyme activity regulation across all known nucleic acid modifying enzymes. Our work also provided a structural rationale for many of the biochemical properties of AID including its unusually low catalytic rate and atypically high nanomolar range binding affinity for its substrate DNA. In Chapter 5, we utilized these structural insights and the combined computational-biochemical approach developed in Chapter 3 to screen a large library of small molecule drug-like compounds against the catalytic pocket of AID. We have identified and functionally tested the first small molecule inhibitors that block the mutagenic activity of AID. We also demonstrated that several of these small molecules can inhibit several of AID’s tumorigenic APOBEC siblings. And finally in Chapter 6, we review several DNA/RNA binding grooves previously discovered on AID and explore their possible binding combinations. We explore how these plastic modes of binding DNA and RNA simultaneously may impact AID function. Finally, we examine proposed AID dimer models bound to DNA/RNA and examine how this may act as an additional regulatory bottleneck of enzymatic activity.

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