Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B

Abstract

APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded (ss)DNA has beneficial functions in immunity and detrimental roles in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Despite numerous structures, mechanisms for global ssDNA recognition and local target sequence selection remain unclear. Here, we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1 and 1.7 angstroms resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring −1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The −1 thymine base fits between flexible loops in a groove that forms upon binding ssDNA, and it makes direct hydrogen bonds with the protein accounting for the strong 5′-TC preference. These studies explain both conserved and unique properties among APOBEC family members, and provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.

Determinants of Substrate Specificity in APOBEC3B

“…26 Both the crystal structures of A3B and our simulations show hydrogen bonds consistently formed between the 3 position NH motif of the T 1 base and the Asp314 sidechain. Interestingly, the rC simulation post pucker change has broken this hydrogen bond and replaced it with one to the sidechain of Asp316.…”

“…25 Finally, a DNA bound crystal structure of A3Bctd was achieved but only after an additional A3A loop 1 swap in addition to the previously used loop 3 truncation and solubilizing mutations. 26 Despite its high value, the information in the A3Bctd crystal structure still leaves some open questions about the native interactions, especially of loop 1, of wild type A3Bctd with its DNA substrate.…”

“…22 Subsequent crystallization of A3 proteins in complex with substrates showed residues on loops 1 and 7 binding to oligonucleotide substrates. 24,26,[36][37][38] Previous experimental studies have explored the binding of A3s and other cytidine deaminases to chemically modified oligonucleotides, such as those with a ribose cytidine (rC) base at the target site, as well as other modifications to the oligonucleotide backbone and base. 20,34,[39][40][41][42][43] These studies have concluded that A3B prefers DNA substrates but can bind and catalyze other substrates with significantly lower activity.…”

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…Although the N-terminal domain (NTD) of A3B is not catalytically active, it does contribute to activity and the CTD alone is 10-fold less active than fl A3B as measured by a mutator assay where A3B is expressed in E. coli or the in vitro activity of maltose binding protein tagged A3B [343,346]. We have little knowledge of why the full length enzyme is more active than the CTD, how fl A3B can access ssDNA in the genome, if it requires cofactors, or how it competes with other ssDNA binding proteins such as replication protein A (RPA) [270,342,344,345]. Although A3A and A3H have been previously characterized biochemically, there is similarly little information on how at a biochemical level these enzymes would access ssDNA in the genome [110,119,174,272,348].…”

“…Despite this understanding of A3B activity at a cellular level, biochemical characterizations have centered on using truncated forms of A3B that contain only the catalytically active C-terminal domain (CTD) [17,175,270,[341][342][343][344][345][346]. A biochemical analysis of full-length (fl) wild-type A3B on substrates relevant to cancer mutagenesis is lacking.…”