Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s

Shurong Hou,Jeong Min Lee,Wazo Myint,Hiroshi Matsuo,Nese Kurt Yilmaz,Celia A Schiffer

doi:10.1016/j.jbc.2021.100909

Abstract

The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.

Highlights

Through deamination, A3s play crucial roles in innate immunity by mutating foreign pathogenic genomes and protecting host cells against retroviruses and retrotransposons [8,9,10,11,12,13,14]
We investigate the structural mechanism of substrate specificity and single-strand DNA (ssDNA)-binding conformation in A3s using a combination of molecular modeling, structural analysis, and parallel molecular dynamics simulations [7, 61,62,63,64,65]
The three A3s investigated, A3A, A3B-C-terminal domain (CTD), and A3GCTD, were modeled and energy minimized with the DNA conformation either in a wrapped (U) or linear (L) shape based on that in the cocrystal structures with A3A and A3G-CTD [47, 48] respectively (Table S1)

Summary

Results

The three A3s investigated, A3A, A3B-CTD, and A3GCTD, were modeled and energy minimized with the DNA conformation either in a wrapped (U) or linear (L) shape based on that in the cocrystal structures with A3A and A3G-CTD [47, 48] respectively (Table S1). The stacking interactions with downstream nucleotides (+1, +2, +3) and hydrogen bonds with the DNA backbone were lost Together these results suggested that A3A prefers binding ssDNA bearing a CTC0 motif in U-shape rather than a linear conformation. In the A3G–DNA cocrystal structure, Trp211 in the 210-PWV-212 insertion stacks with the −30 base and stretches the bound ssDNA into the more extended L-shape This interaction was maintained in the complex during simulations and Trp211 was the residue with the strongest vdW interactions with DNA, indicating that Trp211 is critical for the L binding conformation of ssDNA. The RMSF of C−1 in CCC0 (L) was about the same as T−1 in CTC0 (U) complex (Fig. 2A) Together these molecular interactions reveal how A3A may accommodate and deaminate TC and CC substrate sequence motifs with varied. SsDNA initiated in A3A–CTC0 (L) and A3A– CCC0 (U) complexes underwent conformational changes to

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Discussion

Experimental procedures