Crystal Structure of Cytidine Deaminase Human APOBEC3F Chimeric Catalytic Domain in Complex with DNA

Abstract

APOBEC3F (A3F) demonstrates antivirus activity through its A3F‐CD2 domain catalyzing deamination of cytidine in target motif 5’‐TC in viral cDNA during reverse transcription. But structural basis is unclear. Previously, chimeric A3F‐CD2 (i.e., A3Fc‐CD2, where residues 195—217 of A3F‐CD2 were replaced with residues 197—221 of A3G‐CD2) was found soluble with deamination activity. Here, we report crystal structures of A3Fc‐CD2 in complexes with single‐stranded DNA (5’‐ATTTT5C6A7A8T9T‐3’ and 5’‐ATTTT5C6A7A8C9T‐3’) at resolutions of 1.98 Å and 2.30 Å, respectively. They demonstrate that one molecular DNA binds two A3Fc‐CD2 molecules (monomers A and B) at two sites, distinct from the reported complex structure of human A3F‐CD2 with a 10‐dT DNA. The first site is formed by residues Tyr333, Lys358 and Tyr359 in monomer A, far away from Zn2+ binding motif, interacting with bases dT5, dC6 and dA7 through π‐π stacking and hydrogen bonds. The second site is composed of Trp277, Y307YFW310 near to Zn2+ binding region in monomer B, interacting with base dA8, mainly through π‐π stacking and hydrophobic interactions, determining deamination preference of DNA sequence. Subsequent biochemical investigations further confirm the contribution of these residues to DNA binding and deamination activities. These studies are helpful to understand the mechanism how A3F‐CD2 catalyzes cytidine deamination.

Insights into the Structures and Multimeric Status of APOBEC Proteins Involved in Viral Restriction and Other Cellular Functions

“…For the structural studies of double-domain A3s, a divide-and-conquer approach has been employed in the past, to study individual CD1 or CD2 domains, due to the strong tendency to aggregate for the wild-type full-length proteins. The CD2 domains of A3B [ 177 , 178 ], A3F [ 179 , 180 , 181 , 182 ], and A3G [ 168 , 183 , 184 , 185 , 186 , 187 ] have been determined alone, using X-ray and NMR, and in complex with ssDNA, using crystallography [ 115 , 170 , 188 , 189 , 190 ] or A3F-CD2 with Vif/CBFb partners, using cryoEM [ 191 ], all as monomeric form. The CD1 of A3B [ 163 ] and A3G [ 149 , 192 ] has been determined by X-ray or NMR, which offers some useful information about RNA binding and multimerization for the full-length A3G and A3B.…”

Multifaceted HIV‐1 Vif interactions with human E3 ubiquitin ligase and APOBEC3s

“…The molecular mechanisms of APOBEC3 sequestration by Vif A3 proteins are notoriously difficult to purify in vitro, because A3s usually associate tightly with nucleic acids and form higher ordered oligomers and soluble aggregates. Some soluble constructs of A3F and A3G have been engineered for biochemical and structural studies [47][48][49][50][51][52][53], but the yield of a wild-type protein purified from a recombinant expression system is still limited. The topology of either single-or didomain A3s are highly conserved, composed of five b-strands surrounded by six a-helices.…”

Structural Investigations on the Interactions between Cytidine Deaminase Human APOBEC3G and DNA

“…The structure of inactive pseudo‐catalytic rhesus macaque A3G‐NTD (i.e., rA3G‐NTD) in complex with poly‐dT ssDNA showed that only one deoxythymidine (dT) was observed in a shallow cleft close to the pseudo‐catalytic zinc‐binding motif. In addition, the structure of human A3F‐CD2 (i.e., hA3F‐CD2) in complex with a 10‐dT ssDNA shows that DNA is far away from catalytic zinc‐binding motif, and the structures of chimeric hA3F‐CD2 (i.e., hA3Fc‐CD2) in complex with DNA demonstrate two DNA binding sites on hA3Fc‐CD2 . One is formed by Tyr 333 , Lys 358 and Tyr 359 , the other is composed by conserved residues Trp 277 , Y 307 YFW 310 near to Zn 2+ binding region.…”

“…[27] In contrast, the positiono fD NA in A3F-DNA complex is much far away from the catalytic center, [36] different from those in A3G-PrA,A 3G-PrB, A3A-DNA andA 3B-DNAc omplexes. Moreover,i nA 3G-CD2, the positivelyc harged surface close to the active site is much smaller than those observed in A3A, [34,35] chimericA 3B-CD2, [35] rA3G-CD1 [27] andA 3F-CD2 complexes [36,37] (Figure S7). Thus, A3G-CD2 binds to DNA in am uch weaker manner than A3A, A3B-CD2, A3F-CD1,r A3G-CD1 and A3G-CTD2*, which leads to two DNA binding groovesi nA 3G-CD2.…”

“…In addition, the structure of human A3F-CD2 (i.e.,h A3F-CD2) [36] in complex with a1 0-dT ssDNA shows that DNA is far away from catalytic zinc-bindingm otif, and the structures of chimeric hA3F-CD2( i.e.,h A3Fc-CD2) in complex with DNA demonstrate two DNA binding sites on hA3Fc-CD2. [37] One is formed by Tyr 333 ,L ys 358 and Tyr 359 ,t he other is composed by conserved residues Trp 277 ,Y 307 YFW 310 near to Zn 2 + binding region. Most recently,M aiti, et al,c reateda10site A3G-CD2 variant A3G-CTD2 (i.e.,P 200A,L 234K, N236A, C243A, P247K, Q318K,F 310K, C321A, Q322A, and C356A, in which K247 in loop 3a nd K318 in loop 7e nhance DNA binding) ( Figure S2) with as tronger bindinga ffinity to DNA and higherc atalytic efficiency than its wild-type A3G-CD2.…”