Structure of a Sir2 Substrate, Alba, Reveals a Mechanism for Deacetylation-induced Enhancement of DNA Binding*
Kehao Zhao
,
Xiaomei Chai
and
Ronen Marmorstein
¶
From the
The Wistar Institute and the
Department of Chemistry, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, April 8, 2003
, and in revised form, April 24, 2003.
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ABSTRACT
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The targeted acetylation status of histones and several other
transcriptional regulatory proteins plays an important role in gene
expression, although the mechanism for this is not well understood. As a model
to understand how targeted acetylation may effect transcription, we determined
the x-ray crystal structure of the chromatin protein Alba from
Archaeoglobus fulgidus, a substrate for the Sir2 protein that
deacetylates it at lysine 11 to promote DNA binding by Alba. The structure
reveals a dimer of dimers in which the dimer-dimer interface is stabilized by
several conserved hydrophobic residues as well as the lysine 11 target of
Sir2. We show that, in solution, the mutation of these hydrophobic residues or
lysine 11 disrupts dimer-dimer formation and decreases DNA-binding affinity.
We propose that the in vivo deacetylation of lysine 11 of archaeal
Alba by Sir2 promotes protein oligomerization for optimal DNA binding.
Implications for the mechanism by which histone acetylation modulates gene
expression are discussed.
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INTRODUCTION
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The histone protein building blocks that package DNA into chromatin play
key roles in gene regulation. An octameric core of histone proteins containing
two copies each of histones H2A, H2B, H3, and H4, together with
146 base
pairs of DNA wrapped around these proteins, make up a nucleosome particle
(1). These nucleosome particles
form the higher order chromatin structure that is the scaffold from which gene
regulation must occur. The histone proteins themselves each contain a
globular, highly helical core region that forms the interior of the nucleosome
particle and a highly conserved and considerably more flexible N-terminal tail
region. The histone tails play a very dynamic role in gene regulation by
serving as sites of targeted and extensive post-translation modification,
including acetylation, phosphorylation, and methylation
(2,
3). These histone modifications
also appear to be binding sites for other protein domains
(3). It appears that a specific
histone modification or constellation of modifications leads to distinct
transcriptional events
(46),
although the mechanism by which this occurs is still poorly understood.
Histone H3 methylation at lysine 9 forms one example of how a histone
modification can lead to a downstream transcriptional regulatory event. The
methylation is carried out, at least in part, by the Su(var)3-9 protein, and
the methylated lysine specifically recruits heterochromatin protein 1 (HP1)
though its chromodomain to silence transcription from heterochromatic DNA
(79).
The mechanism by which other histone modifications, including histone
acetylation, modulate transcription is less well understood. Surprisingly,
many proteins initially identified as histone acetyltransferases (HATs), such
as p300/CBP and PCAF, have also been shown to acetylate non-histone
transcription factors, including many transcriptional activators, to promote
gene activation (10). In
addition, histone deacetylases (HDACs) of the Sir2 family have been shown to
deacetylate non-histone transcription factors such as the p53 tumor suppressor
protein (11,
12). Moreover, a Sir2
homologue from the archaea Sulfolobus solfataricus has been reported
to deacetylate a chromatin, non-sequence-specific DNA-binding protein called
Alba (formally known as Sso10b) to promote Alba/DNA association and
transcriptional repression
(13). These additional
acetylation and deacetylation substrates provide convenient models for
understanding the mechanism of acetylation-dependent modulation of protein
activity.
For several reasons, the archaeal Alba protein provides a particularly
interesting model for understanding how the acetylation status of eukaryotic
histones may affect its biological activity. Like the eukaryotic histones,
Alba binds DNA nonspecifically, is acetylated in its N-terminal terminus to
affect its DNA binding and transcriptional regulatory properties, and also
forms higher order structures to bind DNA
(1416).
Interestingly, Alba homologues are also found in eukaryotes
(16), although their greater
sequence divergence than that of the archaeal proteins suggests that they may
have evolved a new function in eukaryotes.
To understand the mechanistic basis for how the acetylation status of Alba
may affect its DNA binding properties, we have determined the high resolution
crystal structure of Alba from the archaea Archaeoglobus fulgidus
(Af-Alba),1 and have
carried out a series of functional studies for correlation with our structural
findings. We find that Af-Alba forms a dimeric structure that is similar to
the dimeric Alba from the archaea S. solfataricus
(15). More interestingly, both
crystal forms of Af-Alba characterized in this study show homologous
dimer-dimer contacts that involve several conserved hydrophobic residues as
well as the lysine 11 substrate for acetylation. Correlating with the
importance of this dimer-dimer interface for DNA binding by Af-Alba and
acetyl-lysine regulation, cross-linking studies reveal the presence of
tetrameric forms of Af-Alba, and mutations of lysine 11 or other hydrophobic
residues at the dimer-dimer interface reduce tetramer formation and DNA
binding activity by Af-Alba. The implications of these studies for
understanding how the acetylation status of eukaryotic histones may affect its
biological function are discussed.
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MATERIALS AND METHODS
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Protein Expression and PurificationThe full-length Alba
gene from A. fulgidus (Af-Alba) was PCR amplified from
A. fulgidus genomic DNA and cloned into the pGEX4T-1 vector to
express the N-terminal glutathione S-transferase (GST) fusion
protein. The plasmid was transformed into the Escherichia coli BL21
(DE3) strain, grown at 37 °C to an absorbance of 0.50.7 at 590 nm,
and induced at 30 °C for 34 h by adding 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside (IPTG). For protein
purification, a cell pellet from a 3-liter growth culture was suspended and
sonicated in a solution containing 20 mM Hepes, pH 7.5, 200
mM NaCl, 10 mM dithiothreitol, and 1 mM PMSF
(buffer A). The supernatant was mixed with about 3 ml of glutathione resin for
2 h at 4 °C, packed into a column, and washed with >40-column volume
buffer A without PMSF. GST-Alba-bound glutathione resin was removed from the
column and treated with thrombin protease at 4 °C for >20 h. The intact
Af-Alba protein containing three additional N-terminal residues remaining from
the thrombin protease cleavage was released from GST in a buffer containing 20
mM Na-citrate, pH 6.0, 100 mM NaCl, 2 mM
dithiothreitol, and 1 mM PMSF (buffer B). The supernatant was
recovered by centrifugation, concentrated to about 2 ml using Millipore
(Amicon) filtration units, and heat treated at 7580 °C for
1020 min to precipitate contaminating proteins. The supernatant was
then recovered by centrifugation and chromatographed on a Superdex-75 gel
filtration column run with buffer B without PMSF. Peak fractions containing
Af-Alba protein eluted in a position close to the 158-kDa globular protein
standard, suggesting an oligomeric state and/or elongated shape for the 10-kDa
Af-Alba protein. Af-Alba was judged to be >95% pure by SDS-PAGE and
concentrated to 2030 mg/ml for storage at -70 °C.
Selenomethionine-derivatized Af-Alba protein was overexpressed from
pGEX4T-1/GST-Alba-transformed bacterial strain B834 (DE3) (Novagen) and grown
in MOPS-based minimal medium as described elsewhere
(17). Substitution mutations
in Af-Alba were generated from the pGEX4T-1 plasmid overexpressing GST-Alba
with sitedirected mutagenesis (QuikChange, Stratagene)
(18), and the respective
mutations were confirmed by DNA sequencing. Selenomethionine-derivatized and
mutant Af-Alba proteins were purified and stored essentially as described for
the unmodified protein.
Crystallization and Structure DeterminationCrystals of
native Af-Alba and selenomethionine-derivatized Af-Alba were grown at room
temperature using the hanging drop vapor diffusion method. Crystals were
obtained in two different forms in the space groups
P43212 with two molecules per asymmetric unit cell and
I212121 with one molecule per asymmetric unit
cell. The P43212 crystal form was obtained by mixing 10
mg/ml protein, 1:1 µl, with a reservoir solution containing 15%
isopropanol, 50 mM sodium cacodylate, pH 6.0, 100 mM
KCl, and 25 mM MgCl2 and equilibrating over 0.5 ml of
reservoir solution. The I212121 crystal form
was obtained by mixing 6 mg/ml protein, 1:1 µl, with a reservoir solution
containing 30% polyethylene glycol (PEG) 400, 100 mM Tris-HCl, pH
8.5, and 200 mM sodium citrate. Both crystal forms grew to a
typical size of 100 x 100 x 30 µm over 6 days. The
P43212 and I212121
crystal forms were transferred to a reservoir solution supplemented with 15%
2-methyl-2,4-pentanediol (MPD) and 40% polyethylene glycol 400 cryoprotecting
reagent, respectively, prior to flash freezing and storage in solid propane
prior to data collection. Native data from both crystal forms and a
three-wavelength (peak, inflection, and remote) multiwavelength anomalous
diffraction (MAD) data set from selenomethionine-derivatized
P43212 crystals were collected on beamline BM19B at the
Advanced Photon Source using an ADSC Quantum-4 CCD detector at 100 K. All data
was processed with the HKL-2000 suite (HKL Research Inc.). Data collection
statistics are presented in Table
I. Four selenium sites were identified in the
P43212 crystal form using CNS
(19) and SOLVE
(20) and confirmed with
cross-difference Fourier maps. Those sites were further refined by CNS and
SOLVE. The map was improved by solvent flattening with the program RESOLVE
(20), and the program O
(21) was used to build
residues 489 of the protein using the selenomethionine sites as guides.
Refinement was carried out using simulated annealing
(22) and torsion angle dynamic
(23) refinement protocols in
CNS with iterative manual adjustments of the model using the program O with
reference to 2Fo -
Fc and Fo -
Fc electron density maps. At 2.65 Å
resolution, a bulk solvent correction
(24) was applied to the data,
individual atomic B-factors were adjusted, and solvent molecules were modeled
into the electron density map. The final model was checked for errors with
composite-simulated annealing omit maps
(25). The
I212121 crystal form was determined by
molecular replacement using the program CNS, with the refined
P43212 crystal form as a search model, and refined to a
resolution of 2.0 Å essentially as described for the
P43212 crystal form. Both structures were refined with
excellent residuals and stereochemistry
(Table I). The
P43212 crystal form was refined to 2.65 Å
resolution with an Rworking and Rfree
of 23.7% and 28.5%, respectively, and the
I212121 form was refined to 2.0 Å
resolution with an Rworking and Rfree
of 22.6% and 25.6%, respectively (Table
I).
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TABLE I Data Collection, phasing, and refinement statistics
SeMet is selenomethionine, MAD is multiwavelength anomalous diffraction,
and FOM is figure of merit.
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Cross-linking and Gel Retardation AssaysThe reagent
ethylene glycol bis[succinimidylsuccinate] (EGS; Pierce) was used for native
and mutant Af-Alba cross-linking studies. Different concentrations of EGS were
mixed with 20 µg of protein in 10 µl of a solution containing 20
mM Hepes, pH 7.5, and 150 mM NaCl and reacted at room
temperature for 30 min. The reaction was stopped by adding 0.35 µl of 1
M Tris-HCl, pH 7.5, and an equal volume of SDS-loading dye and
analyzed on a 420% gradient SDS-PAGE gel. For gel retardation assays
with the native and mutant Af-Alba proteins, 250 ng of pRSET plasmid DNA was
mixed with different concentrations of protein in a solution containing 20
mM MES, pH 6.5, 100 mM NaCl, 1 mM
MgCl2, and 0.1 mg/ml bovine serum albumin and equilibrated at room
temperature for 15 min. The samples were run on 0.7% agarose gels and stained
with ethidium bromide for DNA visualization.
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RESULTS AND DISCUSSION
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Overall Structure of A. fulgidus AlbaThe Af-Alba protein
monomer adopts an elongated shape with dimensions of roughly 23 x 26
x 50 Å with a topology of
1-
1-
2-
2-
3-
4 with each successive
secondary structural element running in opposite directions roughly parallel
to the long dimension of the molecule (Fig
1a). The
3 and
4 strands are particularly
long, extending almost the entire length of the molecule. Both the
P43212 and I212121
crystal forms reveal a 2-fold symmetric Af-Alba dimer burying about 1500
Å2 of solvent-excluded surface in which the molecules
interact side-by side along the long dimension of the monomer and with the
2-fold axis aligned perpendicular to the long dimension of dimer
(Fig. 1b). Residues
from the
2 helix and
3 and
4 strands mediate all of the
dimer contacts (Fig
2a). Not surprisingly, the dimers in both crystal forms
reported here are essentially superimposable, with an r.m.s.d. of 1.3 Å
for C
atoms, and they are also very similar to two different crystal
forms of a previously reported Alba dimer from the archaea S.
solfataricus (15) with an
r.m.s.d. of 3.8 and 3.7 Å for the two crystal forms, respectively.
Notably, nearly all of the residues involved in dimer interactions are
conserved within the Alba proteins, including the Alba homologues from
eukaryotic organisms (Fig. 3),
suggesting that this dimer is a conserved feature of these proteins.

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FIG. 1. Structure of Af-Alba. Structure of the Alba monomer (a),
dimer (b), and tetramer (c) in the crystals.
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FIG. 2. Oligomerization contacts in Af-Alba. a, the interface of
the Alba dimer showing side chains that participate in dimer contacts.
b, the dimer-dimer Alba interface is shown highlighting the side
chain residues that stabilize the interface with a color-coded van der Waals
surface representation.
I212121
and P43212
indicate the tetramer in the two different crystal forms, respectively. The
right panels are close-up views of the dimer-dimer interface.
c, superposition of the Alba dimer-dimer (tetramer) complex in four
different crystal lattices; the two reported here, in space groups
P43212 (cyan) and
I212121 (green), and the previously
reported Alba structures from S. solfataricus, 1HOX
[PDB]
(red)
and 1HOY
[PDB]
(yellow) are shown.
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FIG. 3. Sequence alignment of Alba proteins. An alignment of the archaeal
proteins is shown at the top, and eucaryal proteins are shown and the
bottom. Strictly conserved residues are highlighted in
black, and conservative substitutions are highlighted in gray.
Af-1 and Af-2, A. fulgidus (AF1956 and AF1067, respectively);
Pho, Pyrococcus horikoshii (NP_143671
[GenBank]
); Mth, Methanothemobacter
thermautotrophicus str. Delta H (AAB85958
[GenBank]
); Mja, Methanococcus
jannaschii (Q57665
[GenBank]
); and Sso, Sulfolobus solfataricus (CAA67066
[GenBank]
)
are included in the archaeal protein alignment. Alba proteins from Xenopus
Lavies, Drosophila melanogaster, Arabidopsis thaliana, mouse, and human
are aligned for the eucaryal proteins. Secondary structural elements are
indicated above the aligned sequences. Residues involved in Alba dimer
contacts are highlighted with an open circle, and residues involved
in dimer-dimer contacts are highlighted with a closed circle. The
lysine target for the Sir2 protein is indicated with a cross (+), and
the residues that were mutated for the current study are indicated with an
arrow, which points to the mutated residue.
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Despite the excellent superposition of the four Alba protein dimers, the
tip of the extended
3-
4 hairpin superimposes less well, suggesting
that this region of the protein may be inherently more flexible.
Interestingly, the high mobility group protein IHF uses a similarly extended
-hairpin to bind DNA
(26), suggesting that this
region of Alba may be used for DNA binding.
Higher Order Oligomerization of Af-AlbaInterestingly, both
crystal forms of Af-Alba show very similar dimer-dimer contacts in the crystal
lattice with an r.m.s.d. of 4.3 Å. for the C
atoms between both
tetramers (Fig. 2, b and
d). Like the dimerization interaction, the dimer-dimer
interactions are 2-fold symmetric and, in this case, the interface is formed
primarily by antiparallel interactions between hydrophobic residues in the
1 helices of one subunit of each of the dimers but also involves the
C-terminal tip of the
2 helix. In the P43212
crystal form, the dimer-dimer interface is formed by van der Waals
interactions between Met14 and Met55' (where the
prime designates a residue from the opposing subunit), Leu18 and
Leu18'-Leu21', Phe54 and
Phe54', and an H-bond between Asn25 and
Asn15' with a overall solvent-excluded surface of 4,620
Å2 (Fig.
2c). The dimer-dimer interface of the
I212121 crystal form is somewhat more
intimate, with a solvent-excluded surface of 4,665 Å2. This
dimer-dimer interface shows van der Waals interactions between
Leu18, Leu18'-Leu21', and the
aliphatic region of Asn25' and between Phe54 and
Phe54'. In addition, this interface shows a direct hydrogen
bond between Lys11 (in a loop just N-terminal to the
1
helix) and Glu26' (Fig.
2b). Strikingly similar dimer interfaces are observed in
two different crystal lattices of the Alba structures from S.
solfataricus (15),
although the dimer-dimer interfaces in these structures are more similar to
the P43212 crystal form of Af-Alba
(Fig. 2c). Also
significantly, most of the residues mediating dimer-dimer contacts in Af-Alba
are conserved among the archaeal proteins
(Fig. 3). In particular,
Phe54, Met14, and Lys11 are strictly
conserved, and positions 18 and 21 are invariably hydrophobic. Taken together,
these structural features suggested that the dimer-dimer interactions observed
in the crystals may be physiologically relevant. Noticeably, the conservation
of archaeal residues at the dimer-dimer interface did not extend to the
eukaryotic Alba homologues (Fig.
3), suggesting that the eukaryotic proteins may not form similar
dimer-dimer contacts.
Solution Oligomerization and DNA Binding Properties of Native and
Mutant Af-Alba ProteinsTo test directly the physiological
relevance of the observed dimer-dimer contacts in the crystals, we carried out
mutagenesis of residues that were implicated in playing an important role at
this interface. We chose to mutate Leu18 and Phe54
because they participated in dimer-dimer contacts in both crystal forms; we
also chose to mutate Lys11 because it is the site of deacetylation
by the Sir2 protein, and we entertained the possibility that deacetylation of
this residue may play a modulatory role in dimer-dimer formation. For residues
18 and 54, we chose mutations that would be predicted to be disruptive to the
dimer-dimer interface; therefore, we prepared leucine to arginine (L18R) and
phenylalanine to arginine (F54R) substitutions at residues 18 and 54,
respectively. For lysine 11, we chose both conservative (arginine, K11R) and
non-conservative substitutions for mutagenesis (glutamine, K11Q; and
methionine, K11M). Modeling studies suggested that each of these mutations
would disrupt the Lys11-Glu26' hydrogen bond
revealed to stabilize the hydrogen bond in the
I212121 crystal form. As a control, we also
prepared an asparagine to alanine substitution at residue 10 (N10A). Residue
10 was located at the border of the dimerdimer interface; however, it did not
appear to play a direct role in dimer formation in either of the two crystal
forms of Af-Alba reported here. We therefore expected that the N10A
substitution would behave similarly to the native protein in its ability to
form dimer-dimer contacts. We prepared proteins containing substitution
mutations by site-directed mutagenesis and purified each of the mutants to
homogeneity for analysis in vitro.
To determine the oligomerization states that Af-Alba and each of the
mutants formed in solution, we subjected each of the proteins to cross-linking
with increasing concentrations of the cross-linking reagent EGS. As can be
seen from Fig. 4a, the
native and N10A control proteins form both dimers and tetramers, and, at
higher EGS concentrations, the relative proportions of dimer and tetramer are
comparable. Each of the Af-Alba mutants at the dimer-dimer interface also show
cross-linked dimers and tetramers; but in contrast to the native protein and
the N10A mutant, the amount of cross-linked tetramer is significantly reduced
relative to the amount of respective dimer for each of the mutants. In
addition, the hydrophobic substitution mutants L18R and F18R also form high
order oligomers. Together, the cross-linking data demonstrates that the
Af-Alba mutants at the dimer-dimer interface form less stable tetramers than
the native protein, consistent with the physiological relevance of the Af-Alba
tetramer observed in the crystal lattice.

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FIG. 4. Solution oligomerization and DNA binding properties of Af-Alba.
a, cross-linking of native Af-Alba, a control N10A mutant, and
mutants across the dimer-dimer interface (K11Q, K11M, K11R, L18R, and F54R).
Cross-linking was carried out with 20 µg of protein and increasing
concentrations of the cross-linking reagent EGS and reacted for 30 min.
Lane 2 is Af-Alba without EGS. Reaction products are analyzed on
420% SDS-PAGE gels. b, gel retardation analysis of native
Af-Alba and mutants. 250 ng of plasmid DNA was incubated with increasing
concentration of protein for 15 min followed by electrophoresis on agarose
gels. Lanes 1 and 2 are plasmid and Af-Alba protein alone,
respectively.
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A prediction from the cross-linking studies is that dimerdimer Af-Alba
contacts play a positive regulatory role in DNA binding by Af-Alba, and,
therefore, one would expect that the mutations at the dimer-dimer interface
would decrease DNA binding. To test this directly, we assayed the ability of
Af-Alba and the mutants to band-shift plasmid DNA in agarose gels. We and
others have shown that Alba does not have appreciable affinity for
oligonucleotides (Ref. 15 and
data not shown). Fig
4b shows that 250 ng of plasmid DNA is visibly shifted at
Af-Alba concentrations above 62 ng. At higher concentrations of Af-Alba the
plasmid DNA becomes more retarded until it apparently saturates the plasmid
DNA at about 250 ng of Alba protein. In contrast, each of the proteins with
mutations at the dimer-dimer interface visibly shifts the plasmid DNA at
reproducibly higher protein concentrations of roughly 68-fold. The N10A
mutation, located outside of the dimer-dimer interface and still showing
robust tetramer cross-linking activity
(Fig. 4a), binds DNA
only slightly less well (
2-fold) than the native protein, suggesting that
asparagine 10 of Af-Alba may be involved in direct DNA contact. Taken together
with the structural and cross-linking results, these DNA binding studies imply
that dimer-dimer formation in Af-Alba plays a stimulatory role on DNA binding
by Alba. Moreover, the structural and solution data supports the proposal that
lysine 11 of Alba, the target of deacetylation by the Sir2 protein, plays an
important role in modulating this activity. We presume that the slight
variability of specific dimer-dimer interactions in the various structures
(Fig. 2) and the relatively low
solvent-excluded surface at the dimer-dimer interface relative to the
monomermonomer interface may further facilitate acetylation-dependent
regulation of Alba oligomerization for DNA binding.
Model for DNA Binding by Alba and Its Regulation by
AcetylationThe structural and functional data described above
strongly implies that Af-Alba binds DNA as a dimer of dimers. The high
conservation of residues at the dimer-dimer interface among other archaeal
Alba proteins also implies that all members of the entire family of archaeal
Alba proteins share the same feature (Fig.
3). Notably, many of these residues are not conserved within the
eukaryotic Alba proteins, suggesting that they do not form similar oligomers
for DNA binding (Fig. 3). As
noted previously (15), a DALI
search using the Alba monomer reveals structural similarity to two other known
nucleic acid-binding proteins, translation initiation factor 1 (r.m.s.d. = 2.2
Å) and DNase I (r.m.s.d. = 2.7 Å). The structure of the DNase I in
complex with an 8-base pair DNA duplex suggests where Alba may bind DNA. A
superposition of the protein from the DNase I/DNA complex with one subunit of
the Af-Alba dimer places the DNA on the surface of the protein formed by the
1-
1 and
2-
3 loops as well as the tip of the
3-
4 hairpin (Fig.
5a). This superposition also places the Af-Alba subunit
on the minor groove side of the DNA. Consistent with this general mode of DNA
binding, this surface of the Alba protein contains the most electropositive
charged surface (Fig.
5b), which would nicely complement the electronegative
minor groove of the DNA, and the
1-
1 and
2-
3 loops
contain two of the most highly conserved sequence patches within the archaeal
Alba proteins (Fig. 3). Also
consistent with DNA binding in this mode, only a relatively minor
reorientation of the DNA helical axis of the model would allow each subunit of
the Alba dimer to make symmetric interactions with a B-form DNA duplex
(Fig. 5). Taken together, we
propose that the Alba dimer binds along the minor groove side of DNA, in
contrast to a previous model that places it across the ends of the dimer along
the minor groove with the center of the molecule across the DNA major groove
(15).

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FIG. 5. Model for DNA binding by Alba. a, model for how the Af-Alba
tetramer binds DNA. b, electrostatic surface of the Af-Alba tetramer
modeled on DNA. Blue, red, and white represent the degree of
electropositive, electronegative, and neutral surface potential,
respectively.
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How may the Af-Alba tetramer bind DNA? Upon extending our model for DNA
binding by the Af-Alba dimer to DNA binding by the Af-Alba tetramer, we find
that a sharp DNA bend of about 60° is required to allow each Af-Alba dimer
of the tetramer to bind DNA in the same way for each dimer
(Fig. 5). This proposal is
consistent with the structure of the high mobility group protein IHF bound to
DNA, which also binds essentially B-form DNA as a dimer along the minor groove
and uses extended antiparallel
-hairpin arms, similar to the extended
3-
4 hairpin arms of the Af-Alba dimer, to sharply bend DNA just
outside of the arm regions
(26). The proposal that Alba
may bend DNA is also consistent with its presumed biological role in
facilitating DNA packaging in Archaea
(15) by inducing negative DNA
super coiling (27) without
significant DNA compaction
(14). Interestingly, this
modeling exercise with DNA also places lysine 11, at the edge of the
dimer-dimer interface, in position to also interact with DNA directly. This is
consistent with the slightly greater mutational sensitivity to DNA binding of
Lys11 mutations over the Leu18 and Phe54
mutations (Fig. 4c).
Lysine 11 may therefore play a duel role in facilitating dimer-dimer contacts
as well as direct protein-DNA contacts to enhance Af-Alba affinity for DNA.
The importance of lysine 11 in Af-Alba function is consistent with the
important regulatory role of the deacetylation of this residue by the Sir2
protein. It is possible that Af-Alba forms even higher order structures on
DNA, as suggested by recent studies showing that S. solfataricus Alba
saturates plasmid DNA with a stoichiometry of about 10 bp per protein dimer
(15), a stoichiometry that
would be higher than that predicted from our model for DNA binding. Without
further structural information, it is unclear how this may occur, but it could
involve the incorporation of Alba dimers to bridge contacts between two Alba
tetramers bound to DNA.
Like the archaeal Alba protein, eukaryotic histone proteins are also
acetylated to modulate gene expression. In the case of the histone proteins,
hyperacetylation is generally correlated with gene activation, whereas
hypoacetylation is correlated with repression. The mechanism by which the
acetylation status of histones regulates gene expression is still unclear;
however, several models have been proposed. In one model, acetylation of
lysine residues neutralizes the lysine charge of the lysine residues, thereby
loosing the interaction of the histone tails with DNA and thus increasing DNA
access for the binding of transcription factors to promote transcriptional
activation. A second model argues that the unacetylated histone tails interact
with neighboring nucleosomes in chromatin and that acetylation relieves these
interactions to destabilize repressive higher order chromatin. A third model
argues that histone acetylation is one histone modification among many that
establish a "histone code" to mark histone binding sites for other
transcriptional regulatory proteins to elicit modification-dependant
transcriptional activities
(46).
There have been several studies that support each of these models, and it is
likely that they are not mutually exclusive. The studies we present here
suggest that acetylation-induced regulation of archaeal Alba proteins involve
the modulation of a higher order Alba structure. This would be consistent with
a model in which histone tail acetylation would disrupt higher order chromatin
structure. Confirmation of this model would certainly require further direct
studies involving histone proteins. Nonetheless, the structural and functional
studies presented here suggest that the acetylation status of archaeal Alba
effects its oligomerization status, and it is possible that the evolution to
eukaryotic systems and histone proteins may have maintained this mechanism of
transcriptional regulation.
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FOOTNOTES
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The atomic coordinates and structure factors (code 1NFH and 1NFJ.) have
been deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
* This work was supported by National Institutes of Health Grants GM52880 and
GM60293 (to R. M.) and a grant from the Commonwealth Universal Research
Enhancement Program, Pennsylvania Department of Health (to the Wistar
Institute). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶
To whom correspondence should be addressed. Tel.: 215-898-5006; Fax:
215-898-0381; E-mail:
marmor{at}wistar.upenn.edu.
1 The abbreviations used are: Af-Alba, Alba from Archaeoglobus
fulgidus; GST, glutathione S-transferase; PMSF,
phenylmethylsulfonyl fluoride; MOPS, 4-morpholinepropanesulfonic acid; EGS,
reagent ethylene glycol bis[succinimidylsuccinate]; MES,
4-morpholineethanesulfonic acid. 
 |
ACKNOWLEDGMENTS
|
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We thank Adrienne Clements Egan, Mary Fitzgerald, Hongzhuang Peng, Guoping
Da, Keqin Li, Lan Xu, and Feng Xue for useful discussions and A. Joachimiak,
R. Zhang, N. Duke, and the Structural Biology Center Collaborative Access Team
(SBC-CAT) staff for access to and assistance with the BM19B beamline at
Advanced Photon Source.
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REFERENCES
|
---|
- Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and
Richmond, T. J. (1997) Nature
389,
251-260[CrossRef][Medline]
[Order article via Infotrieve]
- Grant, P. A. (2001) Genome
Biol. 2, Reviews
0003.1-0003.6
- Marmorstein, R. (2001) Nat. Rev. Mol. Cell
Biol. 2,
422-432[CrossRef][Medline]
[Order article via Infotrieve]
- Strahl, B. D., and Allis, C. D. (2000)
Nature 403,
41-45[CrossRef][Medline]
[Order article via Infotrieve]
- Turner, B. M. (2002) Cell
111,
285-291[Medline]
[Order article via Infotrieve]
- Shreiber, S. L., and Bernstein, B. E. (2002)
Cell 111,
771-778[Medline]
[Order article via Infotrieve]
- Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D., and Grewal,
S. I. (2001) Science
292,
110-113[Abstract/Free Full Text]
- Bannister, A. J., Zegerman, P., Partridge, J. F., Miska, E. A.,
Thomas, J. O., Allshire, R. C., and Kouzarides, T. (2001)
Nature 401,
120-124[Medline]
[Order article via Infotrieve]
- Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., and Jenuwein, T.
(2001) Nature
401,
116-120
- Sterner, D. E., and Berger, S. L. (2000)
Microbiol. Mol. Biol. Rev.
64, 435-459[Abstract/Free Full Text]
- Luo, J., Nikolaev, A. Y., Imai, S., Chen, D., Su, F., Shiloh, A.,
Guarente, L., and Gu, W. (2001) Cell
107,
137-148[Medline]
[Order article via Infotrieve]
- Vaziri, H., Dessain, S. K., Ng Eaton, E., Imai, S. I., Frye, R. A.,
Pandita, T. K., Guarente, L., and Weinberg, R. A. (2001)
Cell 107,
149-159[Medline]
[Order article via Infotrieve]
- Bell, S. D., Botting, C. H., Wardleworth, B. N., Jackson, S. P.,
and White, M. F. (2002) Science
296,
148-151[Abstract/Free Full Text]
- Lurz, R., Grote, M., Dijk, J., Reinhardt, R., and Dobrinski, B.
(1986) EMBO J.
5,
3715-3721
- Wardleworth, B. N., Russell, R. J., Bell, S. D., Taylor, G. L., and
White, M. F. (2002) EMBO J.
21,
4654-4662[Abstract/Free Full Text]
- White, M. F., and Bell, S. D. (2002) Trends
Genet. 18,
621-626[CrossRef][Medline]
[Order article via Infotrieve]
- Rojas, J. R., Trievel, R. C., Zhou, J., Mo, Y., Li, X., Berger, S.
L., Allis, D., and Marmorstein, R. (1999)
Nature 401,
93-98[CrossRef][Medline]
[Order article via Infotrieve]
- Papworth, C., Braman, J., and Wright, D. A. (1996)
Strategies 9,
3-4
- Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros,
P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu,
N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L.
(1998) Acta Crystallogr. Sect. D Biol.
Crystallogr. 54,
905-921[CrossRef][Medline]
[Order article via Infotrieve]
- Terwilliger, T. C., and Berendzen, J. (1999)
Acta Crystallogr. Sect. D Biol. Crystallogr.
55, 849-861[CrossRef][Medline]
[Order article via Infotrieve]
- Jones, T. A. (1978) J. Appl.
Crystallogr. 11,
268-272[CrossRef]
- Brunger, A. T., and Krukowski, A. (1990)
Acta Crystallogr. Sect. A
46, 585-593[CrossRef][Medline]
[Order article via Infotrieve]
- Rice, L. M., and Brunger, A. T. (1994)
Proteins 19,
277-290[Medline]
[Order article via Infotrieve]
- Jiang, J. S., and Brunger, A. T. (1994) J.
Mol. Biol. 243,
100-115[CrossRef][Medline]
[Order article via Infotrieve]
- Brunger, A. T., Kuriyan, J., and Karplus, M. (1987)
Science 235,
458-460
- Rice, P. A., Yang, S.-W., Mizuuchi, K., and Nash, H. A.
(1996) Cell
87,
1295-1306[Medline]
[Order article via Infotrieve]
- Xue, H., Guo, R., Wen, Y., Liu, D., and Huang, L.
(2000) J. Bacteriol.
182,
3929-3933[Abstract/Free Full Text]