Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.     

Solution structure of the K50 class homeodomain PITX2 bound to DNA and implications for mutations that cause Rieger syndrome

We have determined the solution structure of a complex containing the K50 class homeodomain Pituitary homeobox protein 2 (PITX2) bound to its consensus DNA site (TAATCC). Previous studies have suggested that residue 50 is an important determinant of differential DNA-binding specificity among homeodomains. Although structures of several homeodomain-DNA complexes have been determined, this is the first structure of a native K50 class homeodomain. The only K50 homeodomain structure determined previously is an X-ray crystal structure of an altered specificity mutant, Engrailed Q50K (EnQ50K). Analysis of the NMR structure of the PITX2 homeodomain indicates that the lysine at position 50 makes contacts with two guanines on the antisense strand of the DNA, adjacent to the TAAT core DNA sequence, consistent with the structure of EnQ50K. Our evidence suggests that this side chain may make fluctuating interactions with the DNA, which is complementary to the crystal data for EnQ50K. There are differences in the tertiary structure between the native K50 structure and that of EnQ50K, which may explain differences in affinity and specificity between these proteins. Mutations in the human PITX2 gene are responsible for Rieger syndrome, an autosomal dominant disorder. Analysis of the residues mutated in Rieger syndrome indicates that many of these residues are involved in DNA binding, while others are involved in formation of the hydrophobic core of the protein. Overall, the role of K50 in homeodomain recognition is further clarified, and the results indicate that native K50 homeodomains may exhibit differences from altered specificity mutants.     

Rapid Identification of Quox-1 Homeodomain DNA-Binding Sequence Using SAAB

Quox-1 is the only gene in the hox family whose expression occurs throughout the development of the central nervous system. Using the Quox-1 homeodomain produced in a bacterial expression system, we were able to identify DNA-binding targets of the Quox-1 protein from a library of randomly generated oligonucleotides by the selection and amplification binding (SAAB) technique. The results indicated that the Quox-1 protein recognizes a new consensus sequence, 5'-CAATC-3', which has not been reported for any other Hox family homeoprotein. In addition, electromobility shift assay further confirmed that the Quox-1 homeoprotein preferentially binds to the 5'-CAATC-3' sequence, but not to the binding sites for other Hox class homeoprotein (TAAT) or NKX class homeoprotein (CAAG). Based on mutation analyses of the DNA sequences, we found that the 5'-CAATC-3' core sequences are required for high affinity binding by the Quox-1 protein. Furthermore, mutation analyses of the Quox-1 homeodomain showed that one of the major determinants participating in recognition of a minor groove is the Gln6 and Thr7 in the N-terminal arm of the homeodomain.     

Two highly related homeodomain proteins, Nkx5-1 and Nkx5-2, display different DNA binding specificities.

The mouse Nkx5-1 and Nkx5-2 genes are related to NK genes in Drosophila and encode proteins with very similar homeodomains. In higher vertebrates Nkx5 genes are specifically expressed in the inner ear. Inactivation of the mouse Nkx5-1 gene by homologous recombination revealed a critical role for the formation of vestibular inner ear structures. Here, we investigated biochemical properties of the proteins encoded by the Nkx5 genes. A similar consensus binding sequence was isolated for both Nkx5 proteins using binding site selection. This sequence is related to target sequences previously identified for other Nkx proteins and contains the conserved homeodomain binding core TAAT. An additional, novel and unrelated high affinity binding sequence could be identified for the Nkx5-2 protein.     

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.     

The three-dimensional structure of the vnd/NK-2 homeodomain-DNA complex by NMR spectroscopy.

The three-dimensional solution structure obtained by NMR of the complex formed between the uniformly singly15N and doubly13C/15N-labeled vnd/NK-2 homeodomain and its consensus 16 base-pair DNA binding sequence was determined. This work was carried out using the accepted repertoire of experiments augmented with a novel implementation of the water flipback technique to enhance signals from exchangeable amide protons. The results using this new technique confirm the existence of hydrogen bonding between the invariant Asn51 and the second adenine of the DNA binding sequence, as seen in crystal structures of other homeodomain-DNA complexes, but never before detected by NMR. Hydrogen bonding by Arg5 and Lys3 in the minor groove of the DNA appears to be responsible for two unusually upfield-shifted ribose H1' resonances. The DNA duplex is nearly straight and its structure is primarily that of B -DNA. A detailed comparison is presented for all available homeodomain-DNA structures including the vnd/NK-2 DNA complex, which demonstrates that homology is maintained in the protein structure, whereas for the orientation of the homeodomain relative to DNA, small but significant variations are observed. Interactions are described involving certain residues in specific positions of the homeodomain, namely Leu7, Thr41, and Gln50 of vnd/NK-2, where single amino acid residue mutations lead to dramatic developmental alterations. The availability of our previously determined three- dimensional structure of the vnd/NK-2 homeodomain in the absence of DNA allows us to assess structural changes in the homeodomain induced by DNA binding.     

Homology modeling based solution structure of Hoxc8-DNA complex: role of context bases outside TAAT stretch

The 3D structure of neither Hoxc8 nor Hoxc8-DNA complex is known. The repressor protein Hoxc8 binds to the TAAT stretch of the promoter of the osteopontin gene and modulates its expression. Over expression of the osteopontin gene is related to diseases like osteoporosis, multiple sclerosis, cancer et cetera. In this paper we have proposed a 3D structure of Hoxc8-DNA complex obtained by Homology modeling and molecular dynamics (MD) simulation in explicit water. The crystal structure (9ant.pdb) of Antennapedia homeodomain in complex with its DNA sequence was chosen as the template based on (i) high sequence identity (85% for the protein and 60% for the DNA) and (ii) the presence of the TAAT stretch in interaction with the protein. The resulting model was refined by MD simulation for 2.0ns in explicit water. This refined model was then characterized in terms of the structural and the interactional features to improve our understanding of the mechanism of Hoxc8-DNA recognition. The interaction pattern shows that the residues Ile(195), Gln(198), and Asn(199), and the bases S2-(4)TAATG(8) are most important for recognition suggesting the stretch TAATG as the "true recognition element" in the present case. A strong and long-lived water bridge connecting Gln(198) and the base of S1-C(7) complementary to S2-G(8) was observed. Our predicted model of Hoxc8-DNA complex provides us with features that are consistent with the available experimental data on Hoxc8 and the general features of other homeodomain-DNA complexes. The predictions based on the model are also amenable to experimental verification.     

Homeodomain binding sites in the 5' flanking region of the Bombyx mori silk fibroin light-chain gene

Multiple A + T-rich stretches in the 5' flanking region of the Bombyx mori fibroin light-chain gene have been shown to bind two Drosophila homeodomain proteins, EVE (even-skipped) and ZEN (zerknüllt), with high affinities. Some of these sites fall into a class that has the established consensus sequence of the binding sites (TCAATTAAAT) for a diverse group of Drosophila homeodomain proteins, while others are quite heterogenous except that they all possess a core TAAT motif. Since clusters of homeodomain binding sites can also be found in the promoters of other silk protein genes, the fibroin gene and the sericin-1 gene, these observations suggest a possible involvement of some homeobox genes in the regulation of a group of silk protein genes.     

Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions

The crystal structure of a complex containing the engrailed homeodomain and a duplex DNA site has been determined at 2.8 A resolution and refined to a crystallographic R factor of 24.4%. In this complex, two separate regions of the 61 amino acid polypeptide contact a TAAT subsite. An N-terminal arm fits into the minor groove, and the side chains of Arg-3 and Arg-5 make contacts near the 5' end of this "core consensus" binding site. An alpha helix fits into the major groove, and the side chains of IIe-47 and Asn-51 contact base pairs near the 3' end of the TAAT site. This "recognition helix" is part of a structurally conserved helix-turn-helix unit, but these helices are longer than the corresponding helices in the lambda repressor, and the relationship between the helix-turn-helix unit and the DNA is significantly different.     

Structural study of homeodomain protein-DNA complexes using a homology modeling approach

The homeodomain is a conserved protein motif that binds to DNA and plays a central role in gene regulation. We use homeodomain as a model system to study the specific interactions between protein and DNA in a complex. Following the fundamental concept of homology modeling, we have developed an algorithm for predicting structures of both protein and DNA using the known structure of a similar complex as the template. The accuracies of the algorithm in predicting the complex structures are evaluated when two of the homeodomain protein-DNA complexes with known structures (antennapedia and MATalpha2) are selected as test systems. This algorithm allows structural studies of homeodomain binds to DNA with different sequences.     

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.     

Homology Modeling Based Solution Structure of Hoxc8-DNA Complex: Role of Context Bases Outside TAAT Stretch

Abstract

The 3D structure of neither Hoxc8 nor Hoxc8-DNA complex is known. The repressor protein Hoxc8 binds to the TAAT stretch of the promoter of the osteopontin gene and modulates its expression. Over expression of the osteopontin gene is related to diseases like osteoporosis, multiple sclerosis, cancer et cetera. In this paper we have proposed a 3D structure of Hoxc8-DNA complex obtained by Homology modeling and molecular dynamics (MD) simulation in explicit water. The crystal structure (9ant.pdb) of Antennapedia homeodomain in complex with its DNA sequence was chosen as the template based on (i) high sequence identity (85% for the protein and 60% for the DNA) and (ii) the presence of the TAAT stretch in interaction with the protein. The resulting model was refined by MD simulation for 2.0ns in explicit water. This refined model was then characterized in terms of the structural and the interactional features to improve our understanding of the mechanism of Hoxc8-DNA recognition. The interaction pattern shows that the residues Ile¹⁹⁵, Gln¹⁹⁸, and Asn¹⁹⁹, and the bases S2-⁴TAATG⁸ are most important for recognition suggesting the stretch TAATG as the ‘true recognition element’ in the present case. A strong and long-lived water bridge connecting Gln¹⁹⁸ and the base of S1-C⁷ complementary to S2-G⁸ was observed. Our predicted model of Hoxc8-DNA complex provides us with features that are consistent with the available experimental data on Hoxc8 and the general features of other homeodomain-DNA complexes. The predictions based on the model are also amenable to experimental verification.     

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.     

Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes

Four molecular dynamics simulation trajectories of complexes between the wild-type or a mutant Antennapedia homeodomain and 2 DNA sequences were generated in order to probe the mechanisms governing the specificity of DNA recognition. The starting point was published affinity measurements showing that a single protein mutation combined with a replacement of 2 base pairs yields a new high-affinity complex, whereas the other combinations, with changes on only 1 macromolecule, exhibited lower affinity. The simulations of the 4 complexes yielded fluctuating networks of interaction. On average, these networks differ significantly, explaining the switch of affinity caused by the alterations in the macromolecules. The network of mostly hydrogen-bonding interactions involving several water molecules, which was suggested both by X-ray and NMR structures of the wild-type homeodomain and its DNA operator sequence, could be reproduced in the trajectory. More interestingly, the high-affinity complex with alterations in both the protein and the DNA yielded again a dynamic but very tight network of intermolecular interactions, however, attributing a significantly stronger role to direct hydrophobic interactions at the expense of water bridges. The other 2 homeodomain-DNA complexes, with only 1 molecule altered, show on average over the trajectories a clearly reduced number of protein-DNA interactions. The observations from these simulations suggest specific experiments and thus close the circle formed by biochemical, structural, and computational studies. The shift from a water-dominated to a more "dry" interface may prove important in the design of proteins binding DNA in a specific manner.