DOI: 10.25549/usctheses-c40-322288
title: Structural studies of APOBEC3 non-catalytic domains

description: The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) is a family of cytidine deaminases that contain homologous cytidine deaminase domains (CD) and catalyze C-to-U mutations on single-stranded DNA (ssDNA) and RNA. A group of members in this family, APOBEC3s (A3s), plays important roles in mammalian innate immune responses. In humans, A3s include seven members consisting of either one or two CDs, with the two domains following the naming convention of CD1 and CD2 when counted from the N- to C-terminus. Current studies have shown that single-domain A3s and CD2s of double-domain A3s are catalytically active, whereas the CD1s are non-catalytic and are proposed to be involved in protein oligomerization, nucleic acid binding, and high-molecular-mass/weight (HMM/HMW) complexes assembly. So far, the structural information of any non-catalytic CD1 domain is limited, mostly due to the difficulties in obtaining the soluble CD1 protein. Therefore, the high-resolution structures of CD1s, and the structure basis for their biological functions remain elusive. ❧ Among all A3s, APOBEC3G (A3G) is reported as the most potent restriction factor of HIV-1. The N-terminal domain of A3G (A3G-CD1) is responsible for oligomerization and nucleic acid binding, both of which are essential for anti-HIV activity. As a countermeasure, HIV-1 viral infectivity factor (Vif) binds A3G-CD1 to mediate A3G degradation. In Chapter 2, we report the crystal structures of a primate A3G-CD1 (rA3G-CD1) alone and in complex with ssDNA. rA3G-CD1 shares a conserved core structure with the previously determined catalytic APOBECs but is unique for an extensively positively-charged surface. Its co-crystal structure with single-stranded DNA (ssDNA) reveals how the conformations of loops and residues surrounding the Zn-coordinated center (Zn-center) change upon DNA binding. We also characterized a dimerization interface of rA3G-CD1, which is important for oligomerization, nucleic acid binding and Vif-mediated degradation. These findings elucidate the molecular basis of antiviral mechanism and HIV-Vif targeting of A3G. ❧ Dysregulation of APOBECs’ catalytic activity may introduce excessive somatic mutations, resulting in genome instability that promotes cancer development and evolution. Recently, the catalytic activity of double-domain APOBEC3B (A3B) has been correlated with kataegis mutational patterns in multiple cancer types. In Chapter 3, we report the crystal structure of a soluble human A3B-CD1 variant and elucidate several structural elements of CD1 involved in catalytic regulation of A3B. Biophysical and biochemical analysis of A3B in human cells revealed that A3B exists in HMW complexes and that enzymatic activity is mostly regulated by the presence of RNA through CD1. Through structural-guided mutational studies, we delineate innate structural elements that promote HMW assembly and facilitate RNA-dependent regulation. Analysis of engineered constructs that dissociate HMW species suggests a potential connection between oligomerization properties and catalytic activity of A3B. Additionally, RNA-dependent attenuation of A3B catalysis is largely alleviated through site-specific, surface charge reduction of CD1. The structural and biochemical insights described here suggest that unique structural features on CD1 regulate the catalytic activity of the A3B through multiple distinct mechanisms.