Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H

Fumiaki Ito,Brett Zirkle,Vagan Arutiunian,Shu-Xing Li,Xiao Xiao,Xiaojiang S. Chen,Aaron Wolfe,Hanjing Yang

doi:10.1038/s41598-018-21955-0

Abstract

APOBEC3H (A3H) is a member of the APOBEC3 subfamily of DNA cytosine deaminases that are important for innate immune defense and have been implicated in cancer biogenesis. To understand the structural basis for A3H biochemical function, we determined a high-resolution structure of human A3H and performed extensive biochemical analysis. The 2.49 Å crystal structure reveals a uniquely long C-terminal helix 6 (h6), a disrupted β5 strand of the canonical five-stranded β-sheet core, and a long loop 1 around the Zn-active center. Mutation of a loop 7 residue, W115, disrupted the RNA-mediated dimerization of A3H yielding an RNA-free monomeric form that still possessed nucleic acid binding and deaminase activity. A3H expressed in HEK293T cells showed RNA dependent HMW complex formation and RNase A-dependent deaminase activity. A3H has a highly positively charged surface surrounding the Zn-active center, and multiple positively charged residues within this charged surface play an important role in the RNA-mediated HMW formation and deaminase inhibition. Furthermore, these positively charged residues affect subcellular localization of A3H between the nucleus and cytosol. Finally, we have identified multiple residues of loop 1 and 7 that contribute to the overall deaminase activity and the methylcytosine selectivity.

Highlights

Among the seven A3H haplotypes, only hap II, V, and VII are reported to effectively restrict Vif-deficient HIV16,18–21, even though hap I was shown to have anti-HIV activity when overexpressed in cell culture[18,19,22,23]
The data reported so far suggest that APOBEC proteins can utilize diverse modes of binding to nucleic acids, all of which fulfill a number of different functions and regulations
The significance of mC deamination by APOBECs has not yet been fully characterized with respect to cellular function; the mC deamination activity associated with Activation-induced deaminase (AID) has been proposed as an alternative demethylation pathway for regulating methylation patterns in genomic DNA of mouse germ cells[44], and for cell reprogramming in induced pluripotent stem cells[45,46,47]

Summary

Introduction

Among the seven A3H haplotypes, only hap II, V, and VII are reported to effectively restrict Vif-deficient HIV16,18–21, even though hap I was shown to have anti-HIV activity when overexpressed in cell culture[18,19,22,23]. The data reported so far suggest that APOBEC proteins can utilize diverse modes of binding to nucleic acids, all of which fulfill a number of different functions and regulations. Mutation of one loop 7 residue, W115, is critical for disrupting the RNA-mediated dimerization of A3H, yielding an RNA-free monomeric protein that still shows binding to nucleic acids and deaminase activity. We show that multiple positively charged residues within this charged surface play an important role for RNA-mediated HMW formation and for RNA-dependent inhibition of deaminase activity. These positively charged residues regulate the subcellular distribution of A3H between nucleus and cytosol. We have identified multiple residues of loop 1 and loop 7 that contribute to overall deaminase activity as well as mC selectivity

Methods

Results

Conclusion