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 cells, affect leaf development due to its aberrant expression in these organs [8]. Additional kn1-like genes (also termed knox genes) have been isolated from maize and other monocot and dicot species Keywords DNA-binding specificity; footprinting; homeodomain; KNOX protein; recognition code Correspondence D. H. Gonzalez, Ca ´ tedra de Biologı ´ a Celular y Molecular, Facultad de Bioquı ´ mica y Ciencias Biolo ´ gicas (UNL), CC 242 Paraje El Pozo, 3000 Santa Fe, Argentina Fax ⁄ Tel: +54 342 4575219 E-mail: dhgonza@fbcb.unl.edu.ar (Received 13 July 2004, revised 31 August 2004, accepted 21 September 2004) doi:10.1111/j.1432-1033.2004.04402.x The interaction of the homeodomain of the sunflower KNOX protein HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl radical footprinting and missing nucleoside experiments. Binding of HAKN1 to different oligonucleotides indicated that HAKN1 prefers the 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- tematic cells in an undifferentiated state. Indeed, over- expression of some class I genes in Arabidopsis and tobacco produces the proliferation of meristems on the surface of leaves. The proteins encoded by knox genes belong to the three-amino-acid loop extension (TALE) superclass. Members of this superclass contain three extra amino acids within the loop connecting helices I and II [22] and are present in several eukaryotic kingdoms, sug- gesting that they represent an early evolutionary acqui- sition. Concerning their interaction with DNA, studies with proteins from barley [23], tobacco [24], rice [25] and maize [26] indicate that they bind related sequences containing a TGAC core (GTCA in the complement- ary strand), considerably different from the sequence TAAT recognized by most homeodomains [27]. Eluci- dating the structural basis for this difference would help to understand at the molecular level how KNOX transcription factors recognize their DNA target site. In this study, we analysed the interaction of the homeodomain of HAKN1, a sunflower class I KNOX protein [28], with DNA. Based on studies of wild-type and mutant forms of the homeodomain, we propose a model for the complex between HAKN1 and its target site. This model must be applicable to all KNOX homeodomains, as important amino acids are con- served within this family. Results Expression and DNA binding analysis of the 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 oligonucleotides (numbers indicated above each lane) as competitors. The sequence of the 7-bp core present in each oligonucleo- tide is shown in (C) for reference. Modifica- tions with respect to BS1 are shown within black boxes. M. F. Tioni et al. KNOX homeodomain–DNA interactions FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 191 bands of similar intensity were observed in this experi- ment. The relative intensity of the low mobility com- plex varied when different protein preparations were used. We speculate that this behavior may arise from aggregation of the protein. Nevertheless, different pro- tein preparations showed the same specificity and affin- ity when considering the amount of bound protein as the sum of both shifted bands. These bands displayed similar footprinting patterns (see below), suggesting that a single HAKN1 homeodomain is bound to DNA in both complexes. This is strengthened by the fact that only monomer–DNA complexes were observed in crosslinking experiments (data not shown). Analysis of the interaction of HAKN1 with different oligonucleotides indicates that modifications in the outermost positions (1 and ⁄ or 7) do not significantly affect binding (Fig. 1A, lanes BS1, 1,7, 7T, 1 and 7C), while certain inner nucleotides, notably those located at positions 4–6, are critical for binding (Fig. 1A, lanes 4, 6A and 5). Regarding position 7, the change of A for T does not seem to affect binding, while the intro- duction of C partially reduces the amount of complex 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 was subjected to hydroxyl radical attack, and free and bound DNA were separated, recovered from the gel and analysed by denaturing polyacrylamide gel electro- phoresis (Fig. 2A). Because the oligonucleotide con- tains two sites in opposite orientation, both strands of the binding site can be observed in a single footprint- ing assay. Analysis of the cleavage patterns indicates that HAKN1 protects six nucleotides from the strand carrying the sequence TGTGACA (hereafter named the top strand). The protected area includes GACA and two adjacent nucleotides (GA) towards the 3¢ end (Fig. 2A). On the bottom strand, the protected region covers two additional nucleotides, AC complementary to GT in TGTGACA (Fig. 2A). For both strands, the highest protection is observed within the GAC core, suggesting that the protein makes closer contacts in this region. This agrees with the important role of these nucleotide positions in determining the binding strength of HAKN1 to DNA shown by electrophoretic mobility shift assays. When the oligonucleotide labeled at its XbaI site (at the opposite 3¢ end) was used, foot- printing patterns were identical to those described above, indicating that HAKN1 makes equivalent contacts with both binding sites present in the 94-bp fragment. Footprinting analysis was also performed with a similar oligonucleotide containing two mutated sites [BS(mut1,7); AGTGACT instead of TGTGACA, mutations underlined). The results obtained were 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). Within the core, modifications to G and A or their complements influence binding more markedly. These results agree with the fact that mutations of these two nucleotides abolish binding of HAKN1 to DNA. On the other hand, because nucleotides at outside posi- tions can be mutated without significant loss in bind- ing efficiency, it can be assumed that they mainly participate in nonspecific contacts, such as those estab- lished with the sugar–phosphate backbone. The results of footprinting and missing nucleoside experiments also indicate that HAKN1 does not make AB Fig. 2. Hydroxyl radical footprinting and interference analysis of HAKN1 binding to DNA. An oligonucleotide containing two HAKN1 binding sites (BS1) in opposite orientations was labeled in the 3¢ end of either strand (HindIII or XbaI sites) and subjected to hydroxyl radical attack either after (A) or before (B) HAKN1 binding. Free (F) and bound (B) DNA were separated and analysed. A portion of the same fragment digested with defined restriction enzymes was used as a standard (S) 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. In the lower part, the sequence of the binding site is shown and the protected positions are indicated in bold and underlined. The GAC (GTC) core that shows the highest protec- tion is shaded. M. F. Tioni et al. KNOX homeodomain–DNA interactions FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 193 symmetrical contacts with its target site. The protein establishes contacts with both strands at the right side of the GAC core, while only one strand seems to be contacted at the left side. This lack of symmetry and the extension of the contacts most probably indicate that only one molecule of HAKN1 is bound at each target site. Binding of HAKN1 single-site mutants to DNA The picture that emerges from our results is that 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 competition experiments (Fig. 3B), where oligonucleo- tides BS(mut1,7) and BS(mut7T) compete more effi- ciently than variants with A [BS1 and BS(mut1)] or C [BS(mut7C)] at this position. Changes at other posi- tions within the target DNA sequence produced sim- ilar effects on binding than with the wild-type protein (Fig. 3). To further explore the hypothesis that residue 50 is oriented towards the 3¢ end of the top strand, we also mutated Ile50 to Lys, present in Drosophila bicoid [33]. I50K–HAKN1 shows a net preference for an oligonucleotide containing the sequence TGACCC [BS(mut7C)] over the original TGACAG, present in AB CD Fig. 3. DNA binding preferences of HAKN1 mutants at position 50. (A) Electrophoretic mobility shift assay of I50S–HAKN1 (30 ng) binding to BS1 and BS(mut1,7). (B) Binding of I50S–HAKN1 to BS(mut1,7) was com- peted with a 100-fold molar excess of oligo- nucleotides with different sequences (depicted in Fig. 1). (C) Binding of I50K– HAKN1 (30 ng) to different oligonucleotides was analysed by an electrophoretic mobility shift assay. In (D), the binding of different amounts (50, 100 and 250 ng) of either HAKN1 or I50K-HAKN1 to oligonucleotides BS1 and BS(mut7C) is shown. Oligonucleo- tide sequences are shown in Fig. 1. 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 is coincident with the one more strongly protected by wild-type HAKN1, but is shorter towards the 3¢ end of the top strand. This result further suggests that Ile50 contacts the nucleotides located 3¢ to the TGAC core, as its replacement by a smaller residue such as Ser allows better access of this region to the modifying agent. Conversely, I50K–HAKN1 shows an extended footprinting pattern towards the 3¢ end of the top strand and the 5¢ end of the bottom strand (Fig. 4B). This agrees with the presence of a larger residue that makes stable contacts with this region of DNA. The interaction of mutants at position 50, and par- ticularly of I50K–HAKN1 with DNA, provides a framework to build a model of HAKN1–DNA inter- actions, taking into account experiments performed with other homeodomains. The protein bicoid, for example, is able to bind the sequence TAATCC that contains the canonical TAAT box [31]. Lys50 of bicoid puta- tively interacts with the CC dinucleotide, as its muta- tion to Gln changes its preference to TAATTG [29]. A reciprocal change, Gln50 to Lys, in engrailed or fushi tarazu shifts sequence preferences from TAATTA or TAATTG to TAATCC [30,34]. We postulate, then, that positioning of the HAKN1 homeodomain along the TGAC core in DNA must be equivalent to that adopted by other homeodomains along the TAAT sequence. The third position of both sequences con- tains an adenine, known to interact with Asn51, AB Fig. 4. Hydroxyl radical footprinting of I50S–HAKN1 (A) and I50K–HAKN1 (B) bound to their preferred binding sites. After binding and hydro- 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 efficiency, suggesting that the mutant homeodomain retains partial specificity. Discrimination at other posi- tions of the bound region is similar to those displayed by the wild-type protein. Although the results obtained do not necessarily indicate a direct role of Lys54 in establishing contacts with DNA, a plausible explan- ation is that this residue interacts with at least one of the members of the CÆG pair at the fourth position of TGAC in the HAKN1–DNA complex. The two leftmost positions of the core interact through the minor groove with the N-terminal arm in most homeodomains [2,31]. In yeast MATa2, for example, the N-terminal arm makes base-specific con- tacts with the first two nucleotides of a TTAC core [5]. Hence, we replaced the N-terminal arm (residues 1–9) of HAKN1 with the same portion of MATa2, to determine if a change in specificity was observed. The resulting protein, Na–HAKN1, showed an overall reduced affinity but the same sequence preferences as HAKN1 (Fig. 6). It is noteworthy that it did not bind oligonucleotide BS(mut4), which contains a TTAC core on the complementary strand. This indicates that the N-terminal arm of MATa2 is not able to interact with DNA within the context of the HAKN1 homeo- domain as it does within MATa2. Poor binding may arise from incorrect folding of the chimeric protein or from the fact that important contacts with DNA are lost upon replacement of the HAKN1 N-terminal arm. In addition to a role of the N-terminal arm in contact- ing the first two amino acids of the core, examination 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 DNA, supporting the hypothesis that Arg55 is involved in base-specific contacts, which are disrupted upon mutation to Lys. Another explanation would be that this change disturbs the overall folding of the homeodomain, but this seems unlikely because several homeodomains, notably MATa2, contain Lys at posi- tion 55. Assuming that the N-terminal arm of MATa2 and Arg55 may be incompatible as both portions may interact with the same positions of the target site, we also constructed a mutant in which the N-terminal arm of MATa2 was inserted into the R55K mutant of HAKN1. This protein was also ineffective in binding to the HAKN1 target site or its variants (Fig. 6). A model for the HAKN1–DNA interaction Based on the analysis of the binding of wild-type and mutant HAKN1 homeodomains to different DNA tar- get sites, we propose a model for the interaction of HAKN1 with DNA. A set of four amino acids, located within helix III of the homeodomain, would make base-specific contacts with defined nucleotides within the tGACAg sequence. Arg55 would establish a pair of hydrogen bonds with positions O6 and N7 of guanine in GACA. As mentioned above, similar inter- actions have been observed in complexes of other homeodomains with DNA [35,36]. Asn51 would inter- act with the first adenine in GACA, also establishing a pair of hydrogen bonds, as in most homeodomain– DNA complexes. The next position (C, or G in the opposite strand) would be contacted by Lys54. Although there is no evidence in the literature about a 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- plexes of extradenticle [35], MATa1 [36] and MATa2 [5] as templates. Figure 7 shows the alignment of helix III of the HAKN1 homeodomain along the major groove of DNA (the MATa1 binding site in this case). Amino acids in red are those present in wild-type HAKN1 that putatively contact the GAC core. Note that Arg55 and Asn51 establish hydrogen bonds with adjacent G and A, respectively. Interestingly, Lys54 also appears making a hydrogen bond with the N7 of an adjacent purine (adenine in this case) present in the complementary strand. A similar contact could be made with a guanine complementary to C in GAC, further suggesting that Lys54 is likely to contact this position. The position of Lys55 in the corresponding mutant is shown in yellow. Clearly, the specific con- tacts made by Arg55 are lost and are replaced by an interaction with the phosphate backbone. Val54 is shown in pink. The shorter side chain and the loss of a hydrogen bond explain the decrease in affinity and relaxed specificity. Finally, the variants at position 50 (Ile in orange, Ser in green and Lys in blue) are also represented. All these residues are oriented towards the 3¢ end of the core and probably establish contacts with the complementary strand. It should be emphasized that the mutagenesis experiments described here do not prove that certain amino acids make base-specific contacts, especially when a new specificity at a defined position was not achieved. The combination of these experiments with the footprinting results and previous M. F. Tioni et al. KNOX homeodomain–DNA interactions 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 HAKN1. They possess Arg at position 54, suggesting that Lys54 in HAKN1 may not be the only means of recognizing the TGAC core. Accordingly, the Bell protein ATH1, which contains Val54, also selects the sequence TGACA from a random population (I. Viola, unpublished results). The presence of Val54 produces, however, a relaxed specificity at the fourth position and reduced affinity within the context of both the HAKN1 (this study) and the ATH1 homeo- domain (I. Viola, unpublished results). MATa1, in turn, binds a completely different sequence (GATGT ⁄ ACATC [44]), indicating that other factors apart from these residues also influence specificity. The GA dinu- cleotide in GATGT is recognized by Arg55 and Asn51 of MATa1, as proposed here for the GA dinucleotide in TGAC. The GT dinucleotide is, in turn, contacted by Met54 and Ile50 through interactions with the com- plementary strand [36]. This means that, in MATa1, positions contacted by residues 55 ⁄ 51 and 54 ⁄ 50 are separated by one additional base pair. This may be originated by the presence of Val47, which binds the nucleotide adjacent to the A recognized by Asn51 in many homeodomains. The above-mentioned TGAC binding proteins (including KNOX proteins) contain Asn47, which may not establish specific contacts with DNA. These differences may also originate changes in the relative orientation of DNA contacting amino acids. The model presented here can also be compared with the structures determined for the TALE proteins 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 present Asn51 is determined by the N-terminal arm and ⁄ or Arg55. The presence of Arg55 determines a G 5¢ to the conserved A, while the N-terminal arm seems to determine a preference for A ⁄ T base pairs. The set of residues present at positions 47, 50 and 54 influence binding preferences at the right side. The putative DNA-contacting amino acids of HAKN1 are also present in all described KNOX pro- teins, indicating that they may all recognize identical or similar sequences. This raises the question of how the specificity of interaction is achieved in vivo, because different KNOX proteins have different functions. A similar paradox has been noted for animal homeo- domains, for which current evidence suggests that spe- cificity arises from the interaction of homeodomain proteins with other factors that somehow influence their DNA binding properties [46]. Plant KNOX pro- teins interact with proteins from the Bell family, which also belong to the TALE superclass [47,48] and bind similar sequences [49]. Chen et al. [49] have shown that potato KNOX and Bell proteins bind two tandem cop- ies of a TGAC motif separated by one additional nuc- leotide. As both types of homedomains seem to establish similar contacts with their target sites (I. Viola, unpublished results), this indicates that the respective recognition helices must lie in an antiparallel orientation within the major groove at opposite sides of the DNA. According to the footprinting data pre- sented here, the central nucleotide pair and the first two pairs of the second TGAC would be contacted by 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 KNOX proteins interact with DNA and how these interactions contribute to the establishment of active transcription complexes that influence defined develop- mental pathways within plant cells. Experimental procedures Cloning, expression and purification of recombinant proteins HAKN1 homeodomain and C-terminal sequences were amplified and cloned in-frame into the EcoRI and PstI sites of the expression vector pMAL-c2 (New England Biolabs, Beverly, MA, USA). Amplifications were performed using Pfu DNA polymerase and oligonucleotides MALN1: 5¢-GC GGAATTCAAAAAGAGAAAGAAAGGG-3¢ and MALC: 5¢-GGCCTGCAGCTAGAGAAGTGAAACATC-3¢ with HAKN1 cDNA [28] as the template. An I50S mutant was constructed using complementary oligonucleotides I50SF and I50SR (5¢-CAACTGGTTC A GCAACCAAAGGAA-3¢ and 5¢-TTCCTTTGGTTGC TGAACCAGTTG-3¢; introduced mutations underlined) together with primers MALC and MALN1, respectively, to amplify partially overlapping N-terminal and C-terminal HAKN1 fragments. The resulting products were mixed in buffer containing 50 mm Tris ⁄ HCl (pH 7.2), 10 mm MgSO 4 , and 0.1 mm dithiothreitol, incubated at 95 °C dur- ing 5 min, and annealed by allowing the solution to cool to 24 °C in approximately 1 h. After this, 0.5 mm of each dNTP and 5 units of the Klenow fragment of E. coli DNA 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 [...]... [53] The corresponding bands were excised from the gel, eluted and analysed on denaturing polyacrylamide gels To determine the position of the footprint within the sequence, a portion of the same fragment was digested with defined restriction enzymes and loaded in the same gel A DNA fragment containing two copies of oligonucleotide BS(mut1,7) was obtained and analysed in a similar way A single copy of. .. R.L.C and D.H.G are members of CONICET; M.F.T and I.L.V are fellows of CONICET Footprinting analysis For the analysis of hydroxyl radical footprinting patterns, a double-stranded oligonucleotide containing the HAKN1 binding site (BS1) with BamHI and EcoRI 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... manufacturers of the pMAL-c2 system The amount of purified protein (> 95% as judged by SDS ⁄ PAGE) was determined as described by Sedmak and Grossberg [52] DNA binding assays For electrophoretic mobility shift assays, aliquots of purified proteins were incubated with double stranded DNA (0.3–0.6 ng, 10 000 c.p.m., labeled with [32P]dATP[aP] by filling-in the 3¢-ends using the Klenow fragment of DNA polymerase)... 94-bp fragment containing 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 fragment of DNA polymerase and [32P]dATP[aP], cleavage with the other enzyme and purification by nondenaturing... (2001) In vitro interactions between barley TALE homeodomain proteins suggest a role for protein protein associations in the regulation of Knox gene function Plant J 27, 13–23 49 Chen H, Banerjee AK & Hannapel DJ (2004) The tandem complex of BEL and KNOX partners is required for transcriptional repression of Ga20ox1 Plant J 38, 276–284 50 Lu Q & Kamps MP (1996) Structural determinants within Pbx1 that... bicoid RNA in organizing the anterior pattern of the Drosophila embryo EMBO J 7, 1749–1756 34 Percival-Smith A, Muller M, Affolter M & Gehring WJ ¨ (1990) The interaction with DNA of wild-type and mutant fushi tarazu homeodomains EMBO J 9, 3967–3974 201 KNOX homeodomain DNA interactions 35 Passner JM, Ryoo HD, Shen L, Mann RS & Aggarwal AK (1999) Structure of a DNA- bound UltrabithoraxExtradenticle homeodomain.. .KNOX homeodomain DNA interactions MAT1 (5¢-AGGGGACATAGATTTACAAAAGAAGCTC GTCAACAA-3¢; first round, MATa2 sequences underlined) or MAT2 (5¢-CGCGAATTCAAGCCGTACAGGGGAC ATAGATTTACA-3¢; second round) together with oligonucleotide MALC (both rounds) All constructions were checked by DNA sequence analysis Expression and purification of the recombinant proteins were carried out as indicated by the manufacturers... within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox -DNA complex Mol Cell Biol 16, 1632–1640 51 Green NC, Rambaldi I, Teakles J & Featherstone MS (1998) A conserved C-terminal domain in PBX increases DNA binding by the PBX homeodomain and is not a primary site of contact for the YPWM motif of HOXA1 J Biol Chem 273, 13273–13279... Binding of HAKN1 to this oligonucleotide (200 000 c.p.m.) was performed at 20 °C in 15 lL of 50 mm 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 to hydroxyl radical cleavage by the addition of 10.5 lL of 200 References 1 Gehring WJ (1987) Homeo boxes in the study of development... homeodomain protein encoded by the hooded gene, k, in barley (Hordeum vulgare) FEBS Lett 408, 25–29 24 Sakamoto T, Kamiya N, Ueguchi-Tanaka M, Iwahori S & Matsuoka M (2001) KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem Genes Dev 15, 581–590 25 Nagasaki H, Sakamoto T, Sato Y & Matsuoka M (2001) Functional analysis of the conserved . 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. Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA Mariana F. Tioni,