No Interaction of Barrier-to-Autointegration Factor (BAF)
with HIV-1 MA, Cone-Rod Homeobox (Crx) or MAN1-C in
Absence of DNA
Ying Huang1, Mengli Cai2, G. Marius Clore2, Robert Craigie1*
1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of
America, 2Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United
States of America
Barrier-to-autointegration factor is a cellular protein that protects retroviral DNA from autointegration. Its cellular role is not
well understood, but genetic studies show that it is essential and depletion or knockout results in lethal nuclear defects. In
addition to binding DNA, BAF interacts with the LEM domain, a domain shared among a family of lamin-associated
polypeptides. BAF has also been reported to interact with several other viral and cellular proteins suggesting that these
interactions may be functionally relevant. We find that, contrary to previous reports, BAF does not interact with HIV-1 MA,
cone-rod homeobox (Crx) or MAN1-C. The reported interactions can be explained by indirect association through DNA
binding and are unlikely to be biologically relevant. A mutation that causes a premature aging syndrome lies on the
previously reported MAN1-C binding surface of BAF. The absence of direct binding of BAF to MAN1-C eliminates disruption
of this interaction as the cause of the premature aging phenotype.
Citation: Huang Y, Cai M, Clore GM, Craigie R (2011) No Interaction of Barrier-to-Autointegration Factor (BAF) with HIV-1 MA, Cone-Rod Homeobox (Crx) or
MAN1-C in Absence of DNA. PLoS ONE 6(9): e25123. doi:10.1371/journal.pone.0025123
Editor: Jean-Luc E. P. H. Darlix, Institut National de la Sante ´ et de la Recherche Me ´dicale, France
Received July 27, 2011; Accepted August 25, 2011; Published September 22, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Intramural Program of NIDDK, National Institutes of Health (NIH), and by the AIDS Targeted Antiviral Program of the
Office of the Director of the NIH (to GMC and RC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Barrier-to-autointegration factor (BAF/BANF1) is cellular
protein that was identified as a factor that blocks autointegration
of retroviral DNA . The role of BAF for the host cell is not well
understood, but knockdown by siRNA or genetic knockout results
in a lethal phenotype that exhibits defects in nuclear morphology
and division [2–5]. BAF is a dimer in solution and the structure of
the dimer has been determined by NMR and X-ray crystallog-
raphy [6,7]. Each subunit of the dimer binds double stranded
DNA non-specifically and BAF therefore bridges together double
stranded DNA molecules ; DNA binding does not induce any
conformational changes in the BAF dimer. At high DNA
concentration bridging results in intermolecular aggregation. At
low DNA concentration, intramolecular bridging results in
compaction of DNA. BAF induced compaction of DNA molecules
can be visualized by total internal reflection fluorescence
microscopy (TIRFM) . DNA stretched out by buffer flow
condenses into a tight ball upon addition of BAF. We hypothesize
that such condensation of retroviral DNA by BAF in the cytoplasm
makes it refractory to autointegration. BAF also interacts with the
LEM domain , a domain that is shared among Lamin-
Associated Polypeptides, Emerin, and Man-1, proteins . An
NMR structure of BAF in complex with the LEM domain of
Emerin reveals that that the BAF dimer binds a single LEM
domain . Thus BAF potentially forms complexes in which the
dimer is associated with a single LEM domain protein. Since each
BAF dimer binds only one LEM domain, each complex contains
only one of the LEM domain proteins and each may differ in their
functional properties. BAF is the primary substrate for phosphor-
ylation by vaccinia-related kinase 1 (VRK1) [13,14]. Vrk1
phoshorylates N-terminal residues of BAF and phosphorylated
BAF no longer binds DNA. Although the interaction of BAF with
DNA and the LEM domain is well understood, much remains to
be learned concerning the essential role of BAF for the cell.
In addition to interacting with DNA and the LEM domain,
BAF has been reported to interact with other cellular and viral
proteins. These include HIV-1 matrix (MA) , cone-rod
homeobox (Crx)  and the C-terminal domain of MAN-1
(MAN1-C) . We sought to probe the structural basis of these
interactions in order to better understand the higher order
complexes that BAF may form in the cell. Contrary to the
previous reports, we found that none of the above proteins interact
with BAF. The reported interactions are likely mediated through
DNA binding. Since any two DNA binding proteins can indirectly
interact through DNA, we conclude that evidence does not
support functionally relevant interaction between BAF and MA,
Crx, or MAN1-C.
Results and Discussion
We used 2D1H-15N heteronuclear single quantum correlation
(HSQC) spectroscopy to monitor interactions between BAF and its
putative binding partners by NMR. In general, each cross-peak in
PLoS ONE | www.plosone.org1September 2011 | Volume 6 | Issue 9 | e25123
the1H-15N HSQC spectrum corresponds to one amino acid in the
15N-labeled protein. Changes in local environment caused by
altered conformation or interaction with other proteins shift or
alter the intensity of related cross-peaks. The HSQC spectrum
therefore represents a fingerprint of the system and can be used to
map binding interfaces. Figure 1 shows the changes in the1H-15N
HSQC spectrum of LEM domains upon interaction with BAF.
Figure 1A shows the1H-15N HSQC spectrum of the LEM domain
of Emerin. Upon incubation with BAF there are major changes in
many of the peaks corresponding to residues that interact with
BAF (Figure 1B). Comparison of the1H-15N HSQC spectrum of
the15N-labeled LEM domain of MAN1 (Figure 1C) with the same
domain bound to BAF (Figure 1D) also reveals considerable
We initially set out to probe the interaction surfaces of BAF and
HIV-1 MA by NMR. Figure 2A shows the
spectrum of15N-labeled MA. To our surprise, addition of BAF to
the MA sample did not result in any changes in the spectrum even
at the protein concentrations used for the NMR measurements.
We conclude that BAF and MA do not directly interact. Then how
might the previously reported interactions  be explained? BAF
and MA both bind DNA and we propose the protein preparations
contained sufficient DNA to allow an apparent interaction
between BAF and MA through DNA binding. Consistent with
this interpretation, the low micromolar range reported apparent
affinity of MA for BAF is similar to the affinity of MA for DNA
. BAF binds DNA more tightly and DNA condensation can
present a kinetic barrier to dissociation . In our hands, both
BAF and MA tend to co-purify with DNA and extensive washing
of columns at high ionic strength is required to remove all traces of
DNA during purification. Some of the previously reported
experiments [15,17] were also carried out with BAF synthesized
in an in vitro coupled transcription/translation system and this
protein will have contained carried over template plasmid DNA.
As expected, when DNA is added to the mixture of BAF and MA
the spectrum radically changes (Figure 2C). We note that the
number of cross-peaks is greatly diminished in contrast with
addition of DNA to MA, alone which shifts and broadens a subset
of peaks without reducing their number . The DNA used for
this experiment was a 16 mer duplex oligonucleotide which
demonstrates that BAF and MA can simultaneously bind to very
Many of the proteins reported to interact with BAF are DNA
binding proteins. We therefore decided to reexamine their binding
in light of our experience with HIV-1 MA. Cone-rod homeobox
(Crx) protein is a transcription factor that was identified as a BAF
interacting protein in a yeast two-hybrid screen . Co-
immunopreciptation and pull-down assays supported the conclu-
sion that BAF and Crx interact directly. Figure 3A and 3B show
the1H-15N HSQC spectrum of15N labeled Crx in the absence
and presence of BAF, respectively. The spectra are identical,
unambiguously indicating that BAF and Crx do not interact
directly. Addition of a 16 mer duplex DNA to the Crx results in a
shift of a subset of the cross-peaks as expected for binding of
DNA by Crx (Figure 3C, compare the spectrum in the presence
of DNA (black) with the superimposed spectrum in the absence of
Figure 1.1H-15N HSQC spectrum of the Emerin (panel A and B) and MAN1 (panel C and D) LEM domains in the absence and
presence of BAF. Spectra were collected on 50 mM15N-labeled Emerin LEM domain (panel A) or 50 mM15N-labeled Emerin LEM domain plus
200 mM BAF2(panel B). Panels C and D show the results of the same experiment substituting 50 mM MAN1 LEM domain for the Emerin LEM domain.
BAF Does Not Bind MA, Crx, or MAN1-C
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DNA (red)). Addition of both DNA and BAF to Crx results in
disappearance of most of the cross-peaks (Figure 3D), indicative of
the formation of large complexes of Crx, BAF and DNA.
MAN1 is an inner nuclear membrane protein that contains a
LEM domain near its N-terminus that binds BAF. The C-terminal
domain of MAN1 (MAN1-C) has been reported to independently
bind BAF  in addition to the transcription factors GCL and
Btf. We labeled MAN1-C with
HSQC spectrum (Figure 4A). Addition of BAF to the MAN1-C
resulted in no significant change in the spectrum (Figure 4B)
demonstrating that these proteins do not interact directly.
Interestingly we find that MAN1-C binds DNA (Figure 4C).
Addition of DNA and BAF to MAN1-C results in additional
disappearance and shifts in peaks demonstrating that BAF and
MAN1-C form a large complex with DNA, although BAF and
MAN1-C do not interact directly.
To confirm that DNA contamination can confound the
interpretation of pull-down assays for protein-protein interactions
we carried out such an assay for BAF and MA interaction in the
absence and presence of DNA. His-tagged BAF was bound to a Ni
chelating sepharose column. MA was then added to the column in
the absence or presence of DNA. After extensive washing BAF was
eluted with imidazole. In the absence of DNA only BAF eluted
from the column (Figure 5, lane 2). However, in the presence of
DNA, MA co-eluted with the BAF (lanes 3 and 4).
The mutation of Ala12Thr in BAF has been identified as the
cause of a human Hereditary Progeroid Syndrome . Ala12 is
surface exposed but does not map to either the DNA or LEM
domain binding surfaces of BAF, so the effects of this mutation are
unlikely to involve disruption of DNA or LEM domain binding. It
was proposed that the mutation might affect the interaction of
BAF with other proteins, its subcellular localization or stability
. Indeed Ala12 lies on the surface of BAF that was implicated
in binding MAN1-C  and disruption of the MAN1-C/BAF
interaction would have been a reasonable candidate for the
primary effect of the mutation. Our finding that MAN1-C does
not interact with BAF eliminates this model. Although we cannot
ignore the possibility that Ala12Thr disrupts an interaction with a
factor yet to be identified, the reduced abundance of BAF in the
mutant cells  suggests a primary effect on protein stability.
Pull-down assays and co-immunoprecipitation are commonly
used to screen for protein-protein interactions because of their
simplicity and convenience. However putative interactions iden-
tified by such assays need to be confirmed and substantiated by
more direct biochemical and biophysical methods. We conclude
that, contrary to previous reports, BAF does not interact with
HIV-1 MA, Crx, or MAN1-C.
15N and recorded the
Materials and Methods
Protein Expression and Purification
Human BAF and the LEM domains from MAN1 (residues 1–
52) and Emerin (residues 1–47) were cloned and purified as
described . MA was purified as described .
MAN1-C (residue 650–911) was subcloned into a modified
pET-32a vector  to form a thioredoxin fusion protein with a
His6 tag and expressed in Escherichia coli strain BL21(DE3)
(Novagen). The construct was verified by DNA sequencing. E. coli
transformed with the MAN1-C vector were grown in minimal
medium with15NH4Cl and glucose as the nitrogen and carbon
sources, respectively. Cells were induced with 1 mM isopropyl D-
thiogalactopyranoside at A6001.0, and harvested by centrifugation
3 h following induction. After harvesting, the cell pellet was
resuspended in 50 ml (per liter of culture) of 50 mM Tris, pH 7.4,
1 M NaCl, 10 mM imidazole, and 1 mM phenylmethylsulfonyl
fluoride. The suspension was lysed by two passages through a
microfluidizer and centrifuged at 10,0006 g for 40 min. The
supernatant fraction was loaded onto a HisTrap HP column (5 ml
per 2 liters of culture; GE Healthcare) equilibrated with 1 M
NaCl, 10 mM imidazole, 20 mM Hepes pH 7.5, 10% glycerol,
free HIV-1 MA, (B) 0.5 mM
unlabeled BAF, (C) 0.5 mM
unlabeled BAF and 2 mM 16 mer DNA.
1H-15N HSQC spectrum of: (A) 0.5 mM
15N-labeled MA plus 1 mM
15N-labeled MA plus 1 mM
BAF Does Not Bind MA, Crx, or MAN1-C
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Figure 3.-1H-15N HSQC spectra of Crx homeodomain. (A) 30 mM free15N-labeled Crx. (B) 30 mM free15N-labeled Crx plus 200 mM BAF2. (C)
30 mM free15N-labeled Crx plus 30 mM 16 mer DNA (black). The spectrum in the absence of DNA is superimposed in red. (D) 30 mM free15N-labeled
Crx plus 200 mM BAF2and 30 mM 16 mer DNA.
Figure 4.1H-15N HSQC spectra of MAN1-C. (A) 50 mM free15N-labeled MAN1-C. (B) 50 mM free15N-labeled MAN1-C plus 200 mM BAF2. (C) 50 mM
free15N-labeled MAN1-C plus 50 mM 16 mer DNA. (D) 50 mM free15N-labeled MAN1-C plus 200 mM BAF2and 50 mM 16 mer DNA.
BAF Does Not Bind MA, Crx, or MAN1-C
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2 mM 2-mercaptoethanol, and the column was extensively
washed with equilibration buffer. The fusion protein was eluted
with a 100 ml gradient of imidazole (25–500 mM) in the same
buffer. The protein was then dialyzed against 20 mM Tris pH 7.5,
200 mM NaCl, and digested with thrombin (10 NIH units/mg of
protein) for 2 hr at room temperature. Thrombin was then
removed by passage over a benzamidine sepharose column. The
cleaved His6-thioredoxin was removed by loading the digested
proteins over a HisTrap HP column. MAN1-C was further
purified by gel filtration on Sephadex-75 gel filtration column (GE
Healthcare) equilibrated with 25 mM potassium phosphate
pH 6.5, 150 mM NaCl, 2 mM 2-mercaptoethanol.
pGEX-4T-2 GST expression vector encoding the Crx homeo-
domain (residues 34–107) plus five N-terminal and nine C-
terminal flanking amino acid residues was a gift from Dr. Shiming
Chen (Washington University in St. Louis), and was expressed in
the E. coli strain BL21(DE3) the same way as described for MAN1-
C above. Cells were lysed the same way as described for MAN1-C
above except that the buffer was 50 mM Tris pH 7.4 containing
0.5 M NaCl. Before eluting the GST fusion protein from the
glutathione Sepharose 4B, the column was washed with at least 20
column volumes phosphate buffered saline (PBS) plus 0.5 M NaCl
until no DNA was present in the eluate. Protein fractions eluted
with 50 mM Tris pH 8 containing 10 mM reduced glutathione
were pooled together and dialyzed against 4L 50 mM Tris pH 7.5
containing 0.5 M NaCl and 2 mM 2-mercaptoethanol. The GST
fusion tag was removed by digestion with thrombin and the Crx
homeodomain was separated by gel filtration on a Sephadex-75
gel column equilibrated with 25 mM potassium phosphate pH 6.5
containing 150 mM NaCl and 2 mM 2-mercaptoethanol.
Protein samples for NMR contained 25 mM potassium
phosphate pH 6.5, 2 mM 2-mercaptoethanol in 95% H2O and
5% D2O with different salt concentrations. Salt concentrations
were 200 mM NaCl for free LEM domains, LEM domain
complexes, Crx, Crx/BAF and Crx/BAF/DNA complexes. The
salt concentration was 150 mM NaCl for MAN1-C, MAN1-C/
BAF and MAN-1C-/BAF/DNA complexes. DNA used for all
NMR experiments was a 16 mer DNA duplex (59 CCAGCA-
CAAACACCTG and its complement).
1H-15N HSQC spectra were recorded at 27uC on Bruker
DRX500 and DRX600 spectrometers equipped with triple
resonance Z gradient cryoprobes. Spectra were processed using
the program NMRPIPE , and analyzed using the program
PIPP . For the LEM domains of Emerin and MAN-1,1H-15N
HSQC spectra were collected on samples of 50 mM free
labeled LEM domain and 50 mM LEM domains plus either
100 mM (50 mM in dimer form) unlabeled BAF or 400 mM
unlabeled BAF. For MAN1-C, spectra were collected on samples
of 50 mM free15N labeled MAN1-C, 50 mM15N labeled MAN1-
C plus 50 mM BAF2or 200 mM BAF2and 50 mM15N labeled
MAN1-C plus 50 mM unlabeled BAF2and 50 mM 16 mer DNA.
For Crx, spectra were collected on samples of 30 mM15N labeled
Crx, 30 mM Crx plus 30 mM or 200 mM unlabeled BAF2and
30 mM15N labeled Crx plus 30 mM BAF2and 30 mM 16 mer
His-tag BAF pull-down assay
Pull-down assays were performed on a 150 ml Ni chelating
sepharose columns equilibrated with 50 mM NaCl, 20 mM Hepes
pH 7.5, 20 mM imidazole, 2 mM 2-mercaptoethanol (binding
buffer). 50 ml of 0.3 mg/ml BAF was then applied in binding buffer.
The column was then washed three times, each with 200 ml of
binding buffer. 50 ml sonicated salmon sperm DNA (0.02 or
0.06 mg/ml) was then applied (when indicated) and the washing
step was repeated. 50 ml of 0.15 mg/ml MA was then applied in
binding buffer and the washing step was repeated. Finally, BAF
was eluted with 70 ml 1 M imidazole pH 7.5. Proteins were
electrophoresed in a 4–12% Bis Tris NuPAGE gel (Invitrogen) and
stained with Coomassie.
Conceived and designed the experiments: MC GMC RC. Performed the
experiments: YH MC. Analyzed the data: MC GMC RC. Contributed
reagents/materials/analysis tools: GMC RC. Wrote the paper: RC MC.
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Figure 5. Co-elution of MA with BAF in pull-down assays in the
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