Abstract
APOBEC3G (A3G) is a potent antiretroviral protein that specifically targets and deaminates cytidine nucleotides to uracil in single stranded DNA (ssDNA) intermediates produced during HIV replication. A3G displays high selectivity for specific polynucleotide contexts targeting 5′‐CC sites for deamination. A3G binds strongly to RNA via the N‐terminal non‐catalytic domain, which is crucial for recruitment of A3G to the virion, yet A3G displays no deamination activity on the cytidines in single stranded RNA. Our recent structure of A3G bound to ssDNA shows the preferred substrate conformation but does not reveal the mechanisms of substrate selection and non‐substrate exclusion. Here, we report NMR and molecular dynamics (MD) simulation based analysis of the interactions between the C‐terminal catalytic domain of A3G (A3Gctd) and multiple substrate and non‐substrate ssDNAs, which are coupled with deamination assays. The extent of interaction with residues located in helix1 and loop1 (T201‐L220) distinguishes substrate ssDNAs from non‐substrate ssDNAs, revealing the difference between catalytic and non‐catalytic ssDNA‐binding modes of A3Gctd. Using fluorine substituted ribonucleotides we show that 2′‐endo sugar pucker of the target cytidine is an essential factor for effective deamination. Furthermore, MD simulations show that ribose cytidine does not stay at catalytic position during 100ns of simulation time whereas deoxy‐ribose cytidine does. Trajectories of the MD simulation suggest that 2′‐hydroxyl group of RNA forms a hydrogen bond with His257 that chelates Zn2+, which results in loss of two hydrogen bonds between the target cytidine and A3Gctd, dislocating cytosine base from the catalytic position. This study provides mechanistic models of the catalytic selectivity of A3G against RNA.Support or Funding InformationInstitute for Molecular Virology Training Program (NIH grant T32 AI83196) University of MinnesotaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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