Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

Abstract

APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd's deamination reactions within a 5=-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5=-CCC substrate over 5=-CCU substrate, which explains why A3G displays almost no processivity within a 5=-CCC motif. In addition, A3Gctd's shortest substrate sequence was found to be a pentanucleotide containing 5=-CCC flanked on both sides by a single nucleotide. A3Gctd as well as fulllength A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo. Human APOBEC3G (A3G) is a member of a family of Zn 2ϩ -dependent polynucleotide cytosine deaminases. This family was named after APOBEC1 (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1) and also includes the antibody gene diversification enzyme AID (activation-induced cytidine deaminase) (reviewed in references 1-5). A3G can restrict HIV-1 replication by packaging into assembling viral particles for delivery to target cells, where it deaminates cytosine to uracil in newly transcribed viral DNA. These cDNA uracils base pair with adenine during plus-strand synthesis and result in G-to-A hypermutation and, in turn, inactivation of the viral genome. A3G has two Zn 2ϩ binding domains that span residues 1 to 196 and 197 to 384, but only the C-terminal domain is catalytically active (6-8). The Nterminal domain interacts with HIV-1 Vif, RNA, and singlestranded DNA (ssDNA) (e.g., see references 7 and 9-11). A3G predominantly deaminates the 3= cytosine (underlined) in a 5=-CCC sequence, although the middle cytosine can also be deaminated in subsequent reactions following deamination of the 3= cytosine (12-16). In longer ssDNA substrates with multiple 5=-CCC sites, A3G deamination exhibits a 3=¡5= spatial preference in vitro (9,17,18). In the present study, we use the catalytic domain of A3G (A3Gctd) to determine kinetic parameters. Our results provide kinetic constants for two independent deaminations within a 5=-CCC sequence, which explain A3G's catalytic site preference for the 3= cytosine. We identify a strong pH dependence of the reaction speed, which implies that a histidine residue is involved in substrate binding. In addition, we identify the shortestlength ssDNA substrate for A3Gctd to be a pentanucleotide. MATERIALS AND METHODSPurification of A3Gctd. The APOBEC3G C-terminal domain (A3Gctd), comprising amino acids 191 to 384, was expressed and purified as previously described (19). Briefly, the glutathione S-transferase (GST)-fused A3Gctd was...

Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity

“…Changing loop 1 in A3A by replacing a single amino acid, H29R, reduced the activity 10-fold compared to WT, with no change in pH dependence (Figure 6D). This is in contrast to the comparable A3G H216R mutant, which unlike WT A3G, exhibits only a small (1.5-fold) increase in deamination activity when the pH is changed from 7.4 to 5.5 117 .…”

A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer

“…A rather strict 5Ј-YYCR motif specificity in the pH 5.5-6.1 range is consistent with the mutation pattern of HIV-1 (9,10,12), which also reflects the favored motif preference for Apo3G acting on HIV-1 cDNA in T cells (5)(6)(7). It has been reported recently that Apo3G has a more modest 4-fold increase in activity at acidic pH 5.5, compared with pH 7.4 (57). Thus, the Apo3A-catalyzed inactivation of HIV-1 in macrophages is fully compatible with its biochemical behavior.…”

Quantitative Analysis of Location‐ and Sequence‐Dependent Deamination by APOBEC3G Using Real‐Time NMR Spectroscopy

“…The real-time NMR method that we used for the analysis of the deamination process has become increasingly popular and has been used by other groups. [18,19] Our real-time NMR method monitors the intensity change of the H5-H6 total correlation spectroscopy (TOCSY) peak of the third cytidine of CCC in real time. Using this method, we previously analyzed the deamination of a hot spot that solely exists in ssDNA.…”