Site-directed mutagenesis and footprinting analysis
of the interaction of the sunflower KNOX protein HAKN1
with DNA
Mariana F. Tioni, Ivana L. Viola, Raquel L. Chan and Daniel H. Gonzalez
Ca
´
tedra de Biologı
´
a Celular y Molecular, Facultad de Bioquı
´
mica y Ciencias Biolo
´
gicas, Universidad Nacional del Litoral, Santa Fe, Argentina
Homeobox genes encode a group of eukaryotic tran-
scription factors generally involved in the regulation of
developmental processes [1]. These genes contain a
region coding for the homeodomain, a 60 amino acid
protein motif that interacts specifically with DNA [2].
The homeodomain folds into a characteristic three-
helix structure. Helices I and II are connected by a
loop, while helices II and III are separated by a turn,
resembling prokaryotic helix-turn-helix transcription
factors. However, unlike helix-turn-helix-containing
proteins, most homeodomains are able to bind DNA
as monomers with high affinity, through interactions
made by helix III (the so-called recognition helix) and
a disordered N-terminal arm located beyond helix I
[3–6].
In plants, the first homeobox was identified in the
maize gene Knotted1 (kn1; [7]). Dominant mutations in
kn1, which is normally active only in meristematic

sequence TGACA (TGTCA), with changes within the GAC core more pro-
foundly affecting the interaction. Footprinting and missing nucleoside
experiments using hydroxyl radical cleavage of DNA showed that HAKN1
interacts with a 6-bp region of the strand carrying the GAC core, covering
the core and nucleotides towards the 3¢ end. On the other strand, protec-
tion was observed along an 8-bp region, comprising two additional nucleo-
tides complementary to those preceding the core. Changes in the residue
present at position 50 produced proteins with different specificities. An
I50S mutant showed a preference for TGACT, while the presence of lysine
shifted the preference to TGACC, suggesting that residue 50 interacts with
nucleotide(s) 3¢ to GAC. Mutation of Lys54 fi Val produced a protein
with reduced affinity and relaxed specificity, able to recognize the sequence
TGAAA, while the conservative change of Arg55 fi Lys completely abol-
ished binding to DNA. Based on these results, we propose a model for the
interaction of HAKN1 with DNA in which helix III of the homeodomain
accommodates along the major groove with Arg55, Asn51, Lys54 and
Ile50, establishing specific contacts with bases of the GACA sequence or
their complements. This model can be extended to other KNOX proteins
given the conservation of these amino acids in all members of the family.
Abbreviations
TALE, three-amino-acid loop extension.
190 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
(reviewed in [9]), indicating that this class of genes
constitutes a family present throughout the plant king-
dom. The knox family of genes can be subdivided into
two classes, I and II, by sequence relatedness and
expression patterns [10]. Based on the expression pat-
terns [11–13], analysis of mutants [14–17] and over-
expression studies [18–21] it was proposed that class
I knox genes are involved in the maintenance of meris-

HAKN1 homeodomain
The homeodomain of the KNOX transcription factor
HAKN1 was expressed in Escherichia coli as a fusion
with the maltose binding protein using vector
pMALc2. The fusion protein was purified by affinity
chromatography in amylose resin and used for DNA–
protein interaction studies. A 24-bp oligonucleotide
(HAKN1 binding site; BS1) containing the sequence
TGT(G ⁄ C)ACA was used as DNA target. This seq-
uence was designed against a compilation of sequences
bound by KNOX transcription factors from different
species, and contains the TGAC (GTCA) core that is
present in all of them.
Figure 1A shows an electrophoretic mobility shift
assay performed with HAKN1 and oligonucleotide
BS1 or variants containing changes at single positions
(sequences shown in the right panel). We have arbi-
trarily numbered from 1 to 7 those positions present in
the strand that contains the central G. Two shifted
B
A
C
Fig. 1. Binding of HAKN1 to different oligo-
nucleotides. (A) Electrophoretic mobility
shift assay performed with 30 ng of HAKN1
and oligonucleotides containing different
variants of the sequence TGT(G ⁄ C)ACA
(numbers indicated above each lane). (B)
Competition assay of HAKN1 binding to BS1
using a 15-fold molar excess of different

formed. Mutations at positions 2 (not shown) and 3
(lane 3) have only a moderate effect. Similar obser-
vations could be made in experiments in which the
binding to oligonucleotide BS1 was competed with a
15-fold molar excess of different oligonucleotides
(Fig. 1B). These results indicate that HAKN1 mainly
recognizes the GAC (GTC) trinucleotide and displays
lower specificity at outer positions. The GAC triplet is
contained within the TGAC sequence, found to be
part of the binding sites of the barley KNOX protein
Hooded [23] and of maize Knotted1 [26]. This element
is also present in the sequence GTNAC, postulated to
be important for the binding of the tobacco protein
NTH15 to DNA [24], provided that N is G or C.
Analysis of DNA binding by hydroxyl radical
footprinting and interference assays
A more detailed picture of the binding of HAKN1 to
its target site was obtained by the analysis of footprint-
ing patterns after cleavage of free and protein-bound
DNA with hydroxyl radicals generated by Fe–EDTA
complexes. For this purpose, a dimer of the corres-
ponding oligonucleotide ligated through its EcoRI
cohesive site was cloned into the BamHI site of pBlue-
script SK
–
. Cleavage with HindIII and XbaI produces
a 94-bp fragment that contains two HAKN1 binding
sites in opposite orientations. After HAKN1 binding
to the 94-bp oligonucleotide, labeled specifically at one
of its 3¢ ends by filling-in the HindIII site, the complex

essentially the same (not shown), suggesting that
HAKN1 contacts the nucleotide adjacent to the GAC
core and its complement on the other strand whether
they are A or T.
Information about the nucleotide positions that
influence binding of HAKN1 to DNA was obtained
from missing nucleoside (interference) experiments.
Here, DNA is treated with hydroxyl radical-generating
agents before protein binding, thus producing a popu-
lation of molecules with single cleavages along the
phosphodiester backbone. This population is incubated
with the protein of interest and subjected to an elec-
trophoretic mobility shift assay from which the free
and bound fractions are recovered. Molecules with
cleavages at positions important for binding are then
under-represented in the bound fraction and, depend-
ing on the binding conditions, over-represented in the
free fraction. Figure 2B shows a missing nucleoside
experiment using HAKN1 and the 94-bp DNA frag-
ment containing two binding sites previously labeled in
one of its 3¢ ends (HindIII or XbaI sites) and treated
with Fe–EDTA. It is noteworthy that there is a good
correlation between the region protected by HAKN1
and the nucleotide positions important for binding.
This means that all nucleotides in the protected area
KNOX homeodomain–DNA interactions M. F. Tioni et al.
192 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
establish contacts that contribute to binding efficiency.
Again, the GAC core seems to be particularly import-
ant, but outside positions are also required (Fig. 2B).

HAKN1 binds an 8-bp region of DNA with a tGACa
(tGTCa) specificity core. An interesting question is
how the HAKN1 homeodomain interacts with this
sequence and which amino acids are involved in
sequence-specific contacts. To answer this, we have
analysed the effect of single-site mutations on HAKN1
binding to TGACA and variants of this sequence. It is
logical to assume that changes in amino acids involved
in the interaction must influence binding efficiency. In
addition, some substitutions may alter binding specific-
ity, indicating the existence of contacts between a given
residue and defined positions within the DNA.
Residue 50 (53 in TALE homeodomains) is usually
involved in determining the different specificities
among related homeodomains [27,29–31]. In homeo-
domains that bind the canonical TAAT sequence,
residue 50 interacts with nucleotides located 3¢ to this
site [27,31]. We reasoned, then, that changing Ile50,
present in HAKN1 and all KNOX proteins, may influ-
ence sequence preferences at external positions of the
core. As a first approach, we mutated Ile50 to Ser, pre-
sent in the yeast TALE protein MATa2 [32]. The ana-
lysis of binding of I50S–HAKN1 to variants of the
HAKN1 binding site indicates a preference for an
oligonucleotide containing the sequence TGACT, while
the wild-type HAKN1 homeodomain binds TGACA
and TGACT with similar efficiency (Fig. 3A). This
suggests that residue 50 interacts with the 3¢ region of
the top strand (and ⁄ or the 5¢ region of the bottom
strand), outside the GAC core. This is also evident in

KNOX homeodomain–DNA interactions M. F. Tioni et al.
194 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
BS1 and BS(mut1) (Fig. 3C). This result confirms that
residue 50 interacts with nucleotides adjacent to the
TGAC core. Binding analysis with different oligonuc-
leotides indicated that I50K–HAKN1 is also able to
interact with oligonucleotide BS(mut6G), that contains
a TGAG core (Fig. 3C). In fact, when higher protein
concentrations were used in the assays, binding to
TGAGAG was considerably better than to TGACAG
(not shown), suggesting that Lys50 may also be able to
contact the fourth position of the core, thus changing
the preference for G. The inclusion of Lys at position
50, in addition to promoting a change in specificity,
resulted in a protein with increased affinity towards its
preferred binding site (Fig. 3D). An additional, fast-
migrating band observed in this experiment is present
in free DNA and may represent noncovalent oligo-
nucleotide dimers interacting through their cohesive
ends. We have observed that the presence of this spe-
cies does not affect the intensity of the shifted band.
The increased affinity dispalyed by I50K–HAKN1
may arise from the fact that lysine is able to establish
hydrogen bonds with DNA, which are more stable
than the van der Waals contacts established by Ile.
The interaction of mutants at position 50 with their
preferred binding sites was also analysed by footprint-
ing experiments. I50S–HAKN1 protects a region cov-
ering five nucleotides of the top strand and six
nucleotides of the bottom strand (Fig. 4A). This region

xyl radical attack, free (F) and bound (B) DNA were separated and analysed. The left and right panels in (A) and (B) represent the top and
bottom strands of the binding site, respectively. A portion of the same fragment digested with defined restriction enzymes was used as a
standard to calculate the position of the footprint. Letters to the right of each panel indicate the DNA sequence (5¢ end in the upper part) of
the corresponding strand in this region. Below the footprints, the sequence of the corresponding binding site is shown and the protected
positions are indicated in bold and underlined.
M. F. Tioni et al. KNOX homeodomain–DNA interactions
FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 195
universally conserved among homeodomains [2,31].
The importance of this interaction is reflected by the
fact that this nucleotide cannot be mutated without a
complete loss of HAKN1 binding. The fourth base in
TAAT is usually recognized by a nonpolar amino acid
(mostly Ile or Val) present at position 47 [2,31].
HAKN1 contains Asn at this position, which may be
too small to establish specific contacts with bases.
Asn47 does not make specific contacts in the homeo-
domain–DNA complexes of MATa2 and extradenticle
[5,35]. Here, we favour the hypothesis that the fourth
position of the core is contacted by Lys54, because the
nucleotide next to that contacted by Asn51 is recog-
nized by residue 54 in other homeodomains (see
below). In support of a prominent role of Lys54, its
mutation to Val produces a significant decrease in
DNA binding (not shown). In addition, K54V–
HAKN1 binds with similar efficiency to sequence vari-
ants containing either A [BS(mut6A)] or C (BS1) at
the fourth position of TGAC, suggesting that it has a
decreased discrimination capacity with respect to wild-
type HAKN1 (Fig. 5). An oligonucleotide containing
TGAG [BS(mut6G)], however, is bound with reduced

Fig. 6. Effect of changes within the N-terminal arm and position 55
on the binding of HAKN1 to oligonucleotides BS1 and BS(mut4).
Binding to oligonucleotides containing the sequences TGACA (BS1)
or TTACA [BS(mut4)] was analysed using 30 ng of proteins HAKN1,
R55K–HAKN1, Na–HAKN1 (a protein containing the N-terminal arm
of MATa2) or R55K–Na–HAKN1 (a protein with both modifications).
A
B
Fig. 5. K54V–HAKN1 shows relaxed specificity. Binding of K54V–
HAKN1 (150 ng) to different oligonucleotides was analysed in an
electrophoretic mobility shift assay (A). (B) Competition of K54V–
HAKN1 binding to BS1 with a 25-fold molar excess of different
oligonucleotides (depicted in Fig. 1).
KNOX homeodomain–DNA interactions M. F. Tioni et al.
196 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
of other homeodomain–DNA complexes suggests the
possibility that Arg55 recognizes the second position
of TGAC. Arg55 participates in binding to G residues
in other homeodomains, such as yeast MATa1
(GATG; [36]) or Drosophila extradenticle (TGAT;
[35]). Consistent with a role in DNA binding, an
Arg55 to Ala mutation completely disrupts the inter-
action of HAKN1 with DNA (not shown). To further
analyse its involvement in base-specific contacts, we
reasoned that a conservative substitution for Lys
would not affect nonspecific interactions (i.e. electro-
static interactions with the phosphate backbone), but
would preclude the establishment of hydrogen bonds
with the guanine base of G. The results shown in
Fig. 6 indicate that R55K–HAKN1 is unable to bind

specific contact made by a lysine at this position, resi-
due 54 interacts with the nucleotide adjacent to that
bound by Asn51 in several homeodomains, for exam-
ple MATa2 (Arg54, TTAC; [5]), TTF1 (Tyr54, CAAG;
[37]), bicoid (Arg54, TAAT or TAAG; [38]) and Hahr1
(Thr54, TAAA, in this case in combination with
Phe47; [39]). Additionally, lysine determines a prefer-
ence for C at an adjacent position when present at
position 50 in bicoid and other mutant homeodomains
(including HAKN1, see above), presumably by inter-
acting with guanine bases through hydrogen bonds as
observed in the Lys50–engrailed crystal structure [40].
Finally, our results also indicate that Ile50 is
involved in establishing a preference for A or T at the
3¢ side of the core. Mutations of this residue to Ser or
Lys were able to confer a new binding specificity to
HAKN1, changing to a net preference for T or C,
respectively. Ile50 is present in MATa1, where it inter-
acts with a TA dinucleotide adjacent to the position
contacted by Met54 [36]. Accordingly, Ile50 may also
be involved in contacts with an adjacent position,
which is protected by HAKN1 in footprinting experi-
ments and interferes with binding when modified by
hydroxyl radical attack.
To examine the consistence of the interactions des-
cribed above, we have constructed a theoretical model
of the HAKN1–DNA complex using the program
swiss-model [41] available in the ExPASy web server.
Different models for wild-type and mutant HAKN1
were obtained using the homeodomain–DNA com-

FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 197
knowledge, however, are highly indicative that this is
the case. Determination of the three-dimensional struc-
ture of the complex will be required to evaluate the
accuracy of the DNA–protein contacts proposed by
this model.
Discussion
In this study, we investigated the interaction of the
homeodomain of the KNOX protein HAKN1 with
DNA. As no structural studies on the interaction of
any KNOX protein with DNA have been reported,
ours constitutes a first approach to understand these
interactions at the molecular level. Electrophoretic
mobility shift assays, footprinting analyses and missing
nucleoside experiments using different binding sites
and mutated proteins allowed us to establish a model
for HAKN1–DNA interaction. This model postulates
that HAKN1 binds to a TGACNN core primarily
through interactions of certain helix III amino acids
(Ile50, Asn51, Lys54 and Arg55) with DNA. This
particular combination of amino acids is present only
in KNOX proteins, indicating that they may have been
selected through evolution to generate a defined specif-
icity. Among them, the incorporation of Ile50 and
Arg55 must have been particularly important. Other
homeodomains that contain Ile50 and Arg55 are those
of the TGIF, Meis and Bell families [22] and yeast
MATa1 [36]. TGIF and Meis proteins bind the
sequence TGTCA (TGACA on the complementary
strand [42,43]), which is identical to that recognized by

extradenticle and PBX1 bound to DNA [35,45]. These
proteins bind the sequence TGAT, with Arg55 and
Asn51 establishing hydrogen bonds with the GA dinu-
cleotide, as proposed here for HAKN1. The initial T is
also contacted by Arg55 through van der Waals inter-
actions in the PBX1 complex [45]. The second T makes
van der Waals contacts with Asn47. As PBX1 and
extradenticle contain Ile54, this situation may resemble
the binding behaviour of K54V–HAKN1, which shows
relaxed specificity at this position.
Our results with HAKN1 clearly support the idea
that there is a general recognition code for homeo-
domains. Accordingly, recognition at the left side of
A
B
Fig. 7. A model for the interaction of HAKN1 with DNA. (A) Dia-
gram of the HAKN1 DNA binding site with the residues putatively
involved in binding each position. (B) Spatial model of the interac-
tion of helix III of wild-type and mutant HAKN1 homeodomains
with DNA. The model was constructed with the program
SWISS-
MODEL [41] using the structures of the DNA complexes of MATa1
(1YRN), extradenticle (1B8I) and MATa2 (1APL) as templates.
Amino acids in red are those present in wild-type HAKN1 that puta-
tively contact the GAC core. Residues at position 50 are: Ile in
orange, Ser in green and Lys in blue. Val54 and Lys55, present in
the mutants, are shown in pink and yellow, respectively.
KNOX homeodomain–DNA interactions M. F. Tioni et al.
198 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
the conserved A that is contacted by the universally

both proteins. This may indicate that some rearrange-
ments may occur upon complex formation by KNOX
and Bell proteins, either before or after binding to
DNA. In the complexes formed by PBX1 and extra-
denticle, the presence of Gly50, which does not contact
DNA, may allow the binding of an additional homeo-
domain in tandem immediately following TGAT
[35,43,45]. The presence of Ile50, that interacts with
nucleotides located at the 3¢ side of the core, may
explain the requirement of a larger distance between
both binding sites in the complexes formed by KNOX
and Bell proteins.
Sequences outside the homeodomain may also influ-
ence the binding properties of the protein. Indeed, a
stretch of 16 amino acids located immediately C-ter-
minal to the homeodomain forms an a-helix that has
been shown to influence the DNA binding affinity of
the PBX1 homeodomain [50,51]. As the protein used
in our assays includes a C-terminal portion, we have
analysed the structure of the region immediately fol-
lowing the HAKN1 homeodomain using several secon-
dary structure prediction programs. We have only
observed a short region (five to eight amino acids
depending on the program) that has a propensity to
form an a-helix. Therefore, we consider it unlikely that
an effect of the C-terminal tail, similar to that
observed with PBX1, occurs in HAKN1 or other
KNOX proteins.
In summary, the results presented here constitute a
framework to understand at the molecular level how

polymerase I were added, and incubation was followed for
1 h at 37 ° C. An aliquot of this reaction was used directly
to amplify the annealed fragments using primers MALN1
and MALC. Mutants I50K, K54V, R55A and R55K were
constructed in a similar way, using oligonucleotides I50KF
(5¢-CAACTGGTTCA
AAAACCAAAGGAA-3¢), I50KR
(5¢-TTCCTTTGGTTT
TTGAACCAGTTG-3¢), K54VF (5¢-
TAAACCARAGG
GTGCGGCAYTGGA-3¢), K54VR (5¢-
TCCARTGCCGC
ACCCTYTGGTTTA-3¢), R55AF (5¢-CA
AAGGAAG
GCGCACTGGAA-3¢), R55AR (5¢-TTCCAG
TGC
GCCTTCCTTTG-3¢), R55KF (5¢-CAAAGGAAGAA
GCACTGGAA-3¢) and R55KR (5¢-TTCCAGTGC
TTCT
TCCTTTG-3¢) to introduce the mutations. The N-terminal
arm of the MATa2 homeodomain (amino acids 1–9) was
introduced into the HAKN1 homeodomain using two
successive rounds of amplification with oligonucleotides
M. F. Tioni et al. KNOX homeodomain–DNA interactions
FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 199
MAT1 (5¢-AGGGGACATAGATTTACAAAAGAAGCTC
GTCAACAA-3¢; first round, MATa2 sequences underlined)
or MAT2 (5¢-CGCGAATTC
AAGCCGTACAGGGGAC
ATAGATTTACA-3¢; second round) together with oligo-

EDTA). The gel was run in 0.5· TBE at 30 mA for 1.5 h
and dried prior to autoradiography.
Footprinting analysis
For the analysis of hydroxyl radical footprinting patterns, a
double-stranded oligonucleotide containing the HAKN1
binding site (BS1) with BamHI and Eco RI compatible
cohesive ends was self-ligated through its EcoRI site to
obtain a dimer and then cloned into the BamHI site of
pBluescript SK
–
. From this clone, a 94-bp fragment con-
taining two HAKN1 binding sites in opposite orientations
was obtained and labeled at one of its 3¢ ends. This was
accomplished by PCR using reverse and universal primers,
followed by cleavage with either HindIII or XbaI (from the
pBluescript polylinker), incubation with the Klenow frag-
ment of DNA polymerase and [
32
P]dATP[aP], cleavage
with the other enzyme and purification by nondenaturing
polyacrylamide gel electrophoresis. Binding of HAKN1 to
this oligonucleotide (200 000 c.p.m.) was performed at
20 °Cin15lLof50mm Tris ⁄ HCl (pH 7.5), 100 mm
NaCl, 10 mm 2-mercaptoethanol, 0.1 mm EDTA,
22 ngÆlL
)1
BSA, 10 ngÆlL
)1
poly(dI-dC) and 800 ng
HAKN1. After 30 min, the binding reaction was subjected

nucleotides containing the different binding sites were
obtained from clones in pBluescript SK
–
as described above
and subjected to hydroxyl radical cleavage [53]. Binding of
HAKN1 to the treated oligonucleotide (200 000 c.p.m.) and
separation of the free and bound fractions by electropho-
retic mobility shift assays were performed as described in
the last section. These fractions were excised from the gel,
eluted and analysed on a denaturing polyacrylamide gel as
described above.
Acknowledgements
This work was supported by grants from CONICET,
ANPCyT and Universidad Nacional del Litoral (Argen-
tina). R.L.C. and D.H.G. are members of CONICET;
M.F.T. and I.L.V. are fellows of CONICET.
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