Engineering the loop region of a homeodomain-leucine zipper protein promotes efficient binding to a monomeric DNA binding site

Plant homeodomain-leucine zipper (HD-Zip) proteins, unlike many animal homeodomains (HDs), are unable to bind DNA as monomers. To investigate the molecular basis of their different behavior, we have constructed chimeras between the HD of the sunflower HD-Zip protein Hahb-4 and that of Drosophila engrailed (EN). Analysis of the interaction of these proteins with the pseudopalindromic Hahb-4 binding site and the monomeric EN binding site suggests that the loop located between helix I and helix II (amino acids 21-28) of EN is enough to confer efficient DNA binding activity to the Hahb-4 HD. Accordingly, the combined mutation of residues 24 and 25 of Hahb-4 to those present in EN (S24R/R25Y) originated an HD able to interact with the EN binding site, while single mutations were ineffective. We have also determined that a protein with the leucine zipper and helix III of Hahb-4 fused to the rest of the EN HD binds to the Hahb-4 pseudopalindomic binding site with increased affinity and shows extended contacts with DNA respective to Hahb-4. We conclude that the loop located between helix I and helix II of the HD must be regarded as one of the segments that contribute to the present-day diversity in the properties of different HDs.     

The role of helix stabilizing residues in GCN4 basic region folding and DNA binding

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil --> helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of alpha-helices, and such N-capping motifs can stabilize alpha-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.     

Characterization of Hoxd1 Protein–DNA-Binding Specificity Using Affinity Chromatography and Random DNA Oligomer Selection

1. Hoxd1 is member of the labial subfamily of Hox genes that has a conserved 60 amino acid homeodomain region. The homeodomain is an important determining factor in the binding of the protein to specific DNA sequence(s). DNA-binding specificity for the Hoxd1 protein has not been determined previously.2. We have employed a rapid affinity chromatography method to determine optimal DNA binding sequences for the 109 amino acid Hoxd1 peptide, comprising the homeodomain and the entire carboxy terminal region of the Hoxd1 protein.3. Labial Hox proteins have intrinsically weak DNA-binding activity that has been attributed to the nonbasic residues at positions 2 and 3 in the N-terminal arm of the homeodomain. The presence of the Hoxd1 carboxy terminal region negated the influence of the nonbasic residues and facilitated Hoxd1 DNA-binding specificity.4. DNA sequences bound to the Hoxd1 peptide-affinity column were separated from a random pool of oligonucleotide sequences by gradient elution and enriched by polymerase chain reaction. Preferred sequences were identified on 5w and 3 of a TAAT core, extending the binding site to T/AT/gTAATTGTA.5. Stability and specificity of optimal DNA-binding sequence for Hoxd1 homeodomain were determined by equilibrium and kinetic studies. Dissociation coefficient constant (K _D) was estimated to be 8.6 × 10^–9 M and the DNA–Hoxd1 homeodomain complex has a half life (t _1/2) of 12.7 min.6. A molecular model of Hoxd1 homeodomain–-DNA interaction based on the X-ray coordinates of Antennapedia homeodomain–DNA complex has revealed novel interactions of key Hoxd1 residues at the protein–DNA interface.     

Structure of homeodomain-leucine zipper/DNA complexes studied using hydroxyl radical cleavage of DNA and methylation interference

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.     

The Yeast a1 and α2 Homeodomain Proteins Do Not Contribute Equally to Heterodimeric DNA Binding

In diploid cells of the yeast Saccharomyces cerevisiae, the α2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the α2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-α2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-α2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the α2 or a1 protein. Interestingly, an α2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of α2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the α2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-α2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.     

The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins.

The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. We compare here the DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity. We further show that amino acid residues within the N-terminal arm are responsible for the sequence specificity differences between the Ultrabithorax and Abdominal-B homeodomains. Similarities and differences at the corresponding positions within the N-terminal arms are conserved in the vertebrate Abdominal-B-like HOM proteins, which play critical roles in limb specifications as well as in regional specification along the anterior-posterior axis. This and other patterns of residue conservation suggest that differential DNA sequence recognition may play a role in HOM protein function in a wide range of organisms.     

Conformational changes of the BS2 operator DNA upon complex formation with the Antennapedia homeodomain studied by NMR with 13C/15N-labeled DNA.

The NMR structures have been determined for a 13C/15N doubly labeled 14 base-pair DNA duplex comprising the BS2 operator sequence both free in solution and in the complex with the Antennapedia homeodomain. The impact of the DNA labeling is assessed from comparison with a previous structure of the same complex that was determined using isotope labeling only for the protein. Differences between the two structure determinations are nearly completely limited to the DNA, which retains the global B -conformation of the free DNA also in the complex. Local protein-induced conformational changes are a narrowing of the minor groove due to the interaction with the N-terminal arm of the homeodomain, and changes of the sugar puckers of the deoxyriboses G5 and C6, which are apparently induced by van der Waals interactions with Tyr25, and with Gln50 and Arg53, respectively. The high conservation of these amino acid residues in homeodomains suggests that protein-induced shifts in some sugar puckers contribute to the affinity of homeodomains to their cognate DNA. The data obtained here with the Antennapedia homeodomain-DNA complex clearly show that nucleic acid isotope-labeling can support detailed conformational characterization of DNA in complexes with proteins, which will be indispensable for structure determinations of complexes containing globally distorted DNA conformations.     

Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain.

In this study, we have investigated the influence of regions outside the DNA-binding domain of the human glucocorticoid receptor on high-affinity DNA binding. We find that the DNA-binding domain shows a 10-fold lower affinity for a palindromic DNA-binding site than the intact receptor. The N-terminal part of the receptor protein does not influence its DNA-binding affinity, while the C-terminal steroid-binding domain increases the DNA-binding affinity of the receptor molecule. It has previously been shown that both the intact glucocorticoid receptor and the glucocorticoid receptor DNA-binding domain bind to a palindromic glucocorticoid response element on DNA as dimers. It is likely that differences in DNA-binding affinity observed result from protein-protein interactions outside the DNA-binding domain between receptor monomers, as has been shown for the estrogen receptor. We have previously identified a segment involved in protein-protein interactions between DNA-binding domains of glucocorticoid receptors. This, in combination with results presented in this study, suggests that there are at least two sites of contact between receptor monomers bound to DNA. We suggest that the interaction between the DNA-binding domains may act primarily to restrict DNA binding to binding sites with appropriate half-site spacing and that additional stability of the receptor dimer is provided by the interactions between the steroid-binding domains.     

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA-binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full-length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA-binding domain of the homeotic proteins.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

DNA-binding activity of amino-terminal domains of the Bacillus subtilis AbrB protein

Two truncated variants of AbrB, comprising either its first 53 (AbrBN53) or first 55 (AbrBN55) amino acid residues, were constructed and purified. Noncovalently linked homodimers of the truncated variants exhibited very weak DNA-binding activity. Cross-linking AbrBN55 dimers into tetramers and higher-order multimers (via disulfide bonding between penultimate cysteine residues) resulted in proteins having DNA-binding affinity comparable to and DNA-binding specificity identical to those of intact, wild-type AbrB. These results indicate that the DNA recognition and specificity determinants of AbrB binding lie solely within its N-terminal amino acid sequence.     

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.     

Helicobacter pylori NikR protein exhibits distinct conformations when bound to different promoters

Helicobacter pylori NikR (HpNikR) is a ribbon-helix-helix (RHH) DNA-binding protein that binds to several different promoter regions. The binding site sequences are not absolutely conserved. The ability of HpNikR to discriminate specific DNA sites resides partly in its nine-amino acid N-terminal arm. Previously, indirect evidence indicated that the arm exists in different conformations when HpNikR is bound to the nixA and ureA promoters. Here, we directly examined HpNikR conformation when it was bound to nixA and ureA DNA fragments by tethering (S)-1{[bis(carboxymethyl)amino]methyl}-2-{4-[(2-bromoacetyl)amino]phenylethyl}(carboxymethyl)amino]acetic acid, iron(III) to different positions in the N-terminal arm and RHH DNA binding domain. Different cleavage patterns at each promoter directly demonstrated that both the RHH domain and the arm adopt different conformations on the nixA and ureA promoters. Additionally, the two RHH domain dimers of the HpNikR tetramer are in distinct conformations at ureA. Site-directed mutagenesis identified an interchain salt bridge (Lys(48)-Glu(47')) in the RHH domain remote from the DNA binding interface that is required for high affinity binding to ureA but not nixA. Finally, DNA affinity measurements of wild-type HpNikR and a salt bridge mutant (K48A) to hybrid nixA-ureA promoters demonstrated that inverted repeat half-sites, spacers, and flanking DNA are all required for sequence-specific DNA binding by HpNikR. Notably, the spacer region made the largest contribution to DNA affinity. HpNikR exhibits a substantially expanded regulon compared with other NikR proteins. The results presented here provide a molecular basis for understanding regulatory network expansion by NikR as well as other prokaryotic regulatory proteins.     

Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was studied in aqueous solution by nuclear magnetic resonance (NMR). Sequence-specific resonance assignments have been obtained for the complete polypeptide chain of 68 amino acid residues. The secondary structure determined from nuclear Overhauser effects (NOE) and information about slowly exchanging amide protons includes three helical segments consisting of the residues 10-21, 28-38 and 42-52, respectively. Combination of the presently available NMR data with computer modeling provided preliminary evidence for the presence of a helix-turn-helix motif in the homeodomain. Near the turn, this supersecondary structure appears to be very similar to the DNA binding site in the 434 and P22 c2 repressors, but both helices in the homeodomain include 2-3 additional residues when compared with these prokaryotic DNA-binding proteins.