Structure of the catalytically active APOBEC3G bound to a DNA oligonucleotide inhibitor reveals tetrahedral geometry of the transition state

Abstract

APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate antiviral immunity. However, the APOBEC3 family has also been linked to many mutational signatures in cancer cells, which has led to an intense interest to develop inhibitors of A3’s catalytic activity as therapeutics as well as tools to study A3’s biochemistry, structure, and cellular function. Recent studies have shown that ssDNA containing 2′-deoxy-zebularine (dZ-ssDNA) is an inhibitor of A3s such as A3A, A3B, and A3G, although the atomic determinants of this activity have remained unknown. To fill this knowledge gap, we determined a 1.5 Å resolution structure of a dZ-ssDNA inhibitor bound to active A3G. The crystal structure revealed that the activated dZ-H2O mimics the transition state by coordinating the active site Zn2+ and engaging in additional stabilizing interactions, such as the one with the catalytic residue E259. Therefore, this structure allowed us to capture a snapshot of the A3’s transition state and suggests that developing transition-state mimicking inhibitors may provide a new opportunity to design more targeted molecules for A3s in the future.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A

“…1g ). This, together with prior work indicating hydration of dZ and its derivatives in structures with CDA, A3A 19-20 and A3G 38-40 , demonstrate a universal mechanism of inhibition 18-19, 38-40 .…”

Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A

“…Very recently, using X-ray crystallography it was revealed that FdZ and dZ form tetrahedral intermediates (4-( R )-hydroxy-3,4-dihydro-2′-deoxy-5-fluorozebularine and 4-( R )-hydroxy-3,4-dihydro-2′-deoxyzebularine) in the enzyme's active site with wild-type A3A 42,43 and catalytically active C-terminal domain of A3G. 59 The position of OH in hydrated FdZ and dZ is the same in the complex with CDA and A3 with carbon C4 having R -stereochemistry. Considering that the nomenclature changes when we replace N3 in 1,3-diazepin-2-one to CH 2 in azepin-2-one (Fig.…”

Synthesis of 1,4-azaphosphinine nucleosides and evaluation as inhibitors of human cytidine deaminase and APOBEC3A

“…We also demonstrated that IIc-and IId-containing DNAs also inhibit fulllength wild-type A3G with similar efficiency to that for the single catalytically active CTD [57,58]. Recently, analysis of crystal structures revealed that both dZ (IIc) and FdZ (IId) form tetrahedral intermediates after hydrolysis of the N3-C4 double bond in the active sites of A3Gctd and A3A [59,60]. The intermediates formed had the same R-stereochemistry at the C4 atom of the nucleobase as previously observed for CDA, and thus confirming the general mechanism of cytosine deamination for A3 and CDA [50,[59][60][61][62][63][64].…”

“…Nucleoside and polynucleotide (A3) CDA share a universal mechanism of target nucleobase engagement, deamination and inhibition [50,[59][60][61][62][63][64]. We have recently demonstrated the first inhibition of A3A-induced mutagenesis in cells using a DNA hairpin carrying FdZ (IId) instead of the target C in the TTC loop [60].…”