Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies
March
Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies
Samantha J. Ziegler 0 1
Chang Liu 0 1
Mark Landau 0 1
Olga Buzovetsky 0 1
Belete A. Desimmie 1
Qi Zhao 0 1
Tomoaki Sasaki 1
Ryan C. Burdick 1
Vinay K. Pathak 1
Karen S. Anderson 0 1
Yong Xiong 0 1
0 Department of Molecular Biophysics and Biochemistry, Yale University , New Haven , Connecticut, United States of America, 2 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 3 Viral Mutation Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute at Frederick , Frederick, Maryland , United States of America
1 Editor: Kefei Yu, Michigan State University , UNITED STATES
-
Data Availability Statement: Our structural data
will be held in the Protein Data Bank (www.rcsb.
org) under the accession code 6BWY. All other
data is contained in the paper and supporting
information files.
Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins
are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to
deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune
response to viral infection by mutating the viral ssDNA. One of the most well-studied human
A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a
specific substrate sequence for catalysisÐfor example, A3G deaminates the third dC in the
CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to
characterize due to poor solution behavior of the full-length protein and loss of DNA affinity
of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the
protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which
we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1
nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the
catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket.
Furthermore, our biochemistry and virology studies provide evidence that the
nucleotidebinding pockets on A3G influence each other in selecting the preferred DNA substrate.
Together, the results provide insights into the mechanism by which A3G selects and
deaminates its preferred substrates and help define how A3 proteins are tailored to recognize
specific DNA sequences. This knowledge contributes to a better understanding of the
mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against
HIV-1.
Science Foundation (NSF-GRFP), and
W81XWH15-1-0290 (to K.S.A from the Department of
Defense). This work was supported in part by the
Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research, and
by Intramural AIDS Targeted Antiviral Program
grant funding to V.K.P. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Introduction
Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G or A3G)
is a human host restriction factor that inhibits human immunodeficiency virus type 1
(HIV1), murine leukemia virus, and equine infectious anemia virus [1±5] primarily through its
deoxycytidine deaminase activity on the viral minus-strand DNA (-ssDNA). A3G is one of the
seven APOBEC3 (A3) proteins that inhibits replication of a diverse set of viruses [4, 6±8]. The
human cytidine deaminase superfamily members have highly conserved protein sequences,
tertiary structural folds, and catalytic mechanisms [
4, 9
]. All A3 proteins and the closely related
activation-induced cytidine deaminase (AID) contain a single catalytically active cytidine
deaminase (CDA) domain. However, some family members, such as A3B, A3D, A3F, and
A3G, have a second pseudocatalytic domain that retains the same tertiary fold, but are not
catalytically active. A3G inhibits viral replication by preferentially deaminating dC to dU in the
viral -ssDNA during reverse transcription [7, 10±12]. Specific ªhotspotº sequences of DNA are
targeted for deamination, with the highest preference for the 5'-CCCA sequence (where the
deaminated C is underlined) in the case of A3G [
2, 13
]. Deamination of the -ssDNA by A3G
leads to extensive G-to-A hypermutation in the viral genome, ultimately eliminating viral
infectivity [
1, 5, 13
]. Some studies suggest that mutations induced by A3G are occasionally
sub-lethal, facilitating viral evolution and allowing the virus to develop drug resistance [
3, 14
].
However, a recent study concluded that hypermutation is almost always a lethal event and
makes little or no contribution to viral genetic variation [15]. This underlines the importance
of understanding the mechanism by which A3G induces mutations in the HIV-1 genome [
3,
16
]. While the A3s (...truncated)