A Hydrodynamic Analysis of APOBEC3G Reveals a Monomer−Dimer−Tetramer Self-Association That Has Implications for Anti-HIV Function

Salter, Jason D.; Krucinska, J.; Raina, Jay; Smith, Harold C.; Wedekind, Joseph E.

doi:10.1021/bi901642c

Cited by 39 publications

(60 citation statements)

References 24 publications

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“…Although these data affirmed the assertion that homomultimerization of A3G was not necessary for ssDNA binding and deaminase activity, what remains is that native A3G forms stable dimers (20) and that monomers are rare biological isolates.…”

supporting

confidence: 54%

“…An elongated homodimer of A3G involving a dimer interface in the C termini was predicted by small angle x-ray scattering and corroborated through immunoprecipitation studies of A3G deletion constructs (18,19). Analytical ultracentrifugation of native, full-length A3G also showed that in the absence of nucleic acids, A3G was predominantly a homodimer (20). At low A3G concentrations, less than 5% of the protein was monomeric.…”

mentioning

confidence: 67%

“…In this regard, unlike native A3G homodimers whose assembly is not dependent on nucleic acid binding (18 -20), the formation of tetramers and higher-order complexes of A3G may be enhanced (bridged) by nucleic acids binding. This possibility is supported by analytical ultracentrifugation data showing that nucleic acid-depleted A3G protein concentration had to be high in order for homotetramers to form (20).…”

Section: Discussionmentioning

confidence: 90%

“…Previous analytical ultracentrifugation showed that nucleic acid-depleted A3G protein-protein dimer had 5.6 S (20). Given that the ssDNA molecular mass was 9.2 kDa, the C1 complex was likely to be a dimer of A3G plus one or two molecules of ssDNA.…”

Section: Evaluation Of A3g Composition Of 58 S and 16 S Complexes-mentioning

confidence: 96%

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Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes

McDougall¹,

Okany²,

Smith

2011

Journal of Biological Chemistry

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APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4°C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37°C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription.APOBEC3G is a cytidine deaminase whose expression has been implicated in host defense and conditionally in viral resistance (1). The APOBEC 4 protein family includes APOBEC1 (after which the family is named), activation-induced deaminase, APOBEC2, APOBEC3A-H, and APOBEC4 (2-5). The catalytic activity of these enzymes involves hydrolytic deamination of cytidine to form uridine or deoxycytidine to form deoxyuridine in single-stranded RNA or single-stranded DNA. There is considerable interest in this class of proteins because they are capable of mutagenic activity and epigenetic regulation of protein variant expression that can influence tissue functions and disease progression (4, 6).APOBEC proteins are characterized by a helix-strand-helix supersecondary structure containing conserved residues (His/ Cys)-Xaa-Glu-Xaa 25-30 Pro-Cys-Xaa-Xaa-Cys that are integral to the function of the zinc-dependent deaminase domain or ZDD (5). Several of the APOBEC proteins, such as APOBEC1 and activation-induced deaminase, contain a single ZDD that was responsible for catalytic activity. Other family members, such as APOBEC3B and APOBEC3G, contain multiple ZDDs within a single molecule where both domains or a single domain were catalytically active (2, 3).The N-terminal and C-terminal ZDD in A3G were required for nucleic acid-dependent higher-order oligomerization as well as many other interactions necessary for antiviral activity and wild type levels of deaminase activity (7-11). In isolation, the N-terminal ZDD could not support RNA or ssDNA binding (12), but the C-terminal half of A3G supported a modest level of deaminase activity (12-16).The crystal structure of full-length APOBEC2 and lower resolution small angle x-ray scattering structure of native, fulllength A3G revealed ...

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supporting

confidence: 54%

mentioning

confidence: 67%

Section: Discussionmentioning

confidence: 90%

Section: Evaluation Of A3g Composition Of 58 S and 16 S Complexes-mentioning

confidence: 96%

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Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes

McDougall¹,

Okany²,

Smith

2011

Journal of Biological Chemistry

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“…This co-factor is coordinated by the evolutionarily conserved motif (H/C)…CXXC, which constitutes a key signature sequence for members of the Cytidine Deaminase (CDA) superfamily. In addition, a majority of nucleotide and editing deaminases exist in cells as functional multimers, generally homodimers or homotetramers (30,31), formed by identical subunits. Regardless of their multimeric state, one Zn 2ϩ ion is bound per subunit, and it has been suggested that in many cases each active site acts independently.…”

Section: Discussionmentioning

confidence: 99%

A Single Zinc Ion Is Sufficient for an Active Trypanosoma brucei tRNA Editing Deaminase

Spears

Rubio

Gaston

et al. 2011

Journal of Biological Chemistry

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Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn 2؉ . The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn 2؉ per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn 2؉ -coordinating cysteines in TbADAT2 yields a functional enzyme with a singlebound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn 2؉ . These observations raise the possibility of an unusual Zn 2؉ coordination interface with important implications for the function and evolution of editing deaminases.

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The multifaceted roles of RNA binding in APOBEC cytidine deaminase functions

et al. 2014

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Cytidine deaminases have important roles in the regulation of nucleoside/deoxynucleoside pools for DNA and RNA synthesis. The APOBEC family of cytidine deaminases (named after the first member of the family that was described, Apolipoprotein B mRNA Editing Catalytic Subunit 1, a.k.a. APOBEC1 or A1) is a fascinating group of mutagenic proteins that use RNA and single stranded DNA (ssDNA) as substrates for their cytidine or deoxycytidine deaminase activities. APOBEC proteins and base-modification nucleic acid editing have been the subject of numerous publications, reviews and speculation. These proteins play diverse roles in host cell defense, protecting cells from invading genetic material, enabling the acquired immune response to antigens and changing protein expression at the level of the genetic code in mRNA or DNA. The amazing power these proteins have for interphase cell functions relies on structural and biochemical properties that are beginning to be understood. At the same time, the substrate selectivity of each member in the family and their regulation remains to be elucidated. This review of the APOBEC family will focus on an open question in regulation, namely what role the interactions of these proteins with RNA have in editing substrate recognition or allosteric regulation of DNA mutagenic and host defense activities.

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A Hydrodynamic Analysis of APOBEC3G Reveals a Monomer−Dimer−Tetramer Self-Association That Has Implications for Anti-HIV Function

Cited by 39 publications

References 24 publications

Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes

Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes

A Single Zinc Ion Is Sufficient for an Active Trypanosoma brucei tRNA Editing Deaminase

The multifaceted roles of RNA binding in APOBEC cytidine deaminase functions

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