Molecular dynamics analysis of the engrailed homeodomain-DNA recognition

Molecular dynamics (MD) simulations were performed for investigating the role of Gln50 in the engrailed homeodomain-DNA recognition. Employing the crystal structure of free engrailed homeodomain and homeodomain-DNA complex as a starting structure, we carried out MD simulations of: (i) the complex between engrailed homeodomain and a 20 base-pair DNA containing TAATTA core sequence; (ii) the free engrailed homeodomain. The simulations show that homeodomain flexibility does not depend on its ligation state. The engrailed homeodomain shows similar flexibility, and the recognition helix-3 shows very similar characteristic of high rigidity and limited conformational space in two complexation states. At the same time, DNA structure has also no obvious conformational fluctuations. These results preclude the possibility of the side chain of Gln50 forming direct hydrogen bonds to the core DNA bases. MD simulations confirm a few well-conserved sites for water-mediated hydrogen bonds from protein to DNA are occupied by water molecules, and Gln50 interacts with corresponding core DNA bases through water-mediated hydrogen bonds. So Gln50 plays a relatively modest role in determining the affinity and specificity of the engrailed homeodomain. In addition, the electrostatic interaction between homeodomain and phosphate backbone of the DNA is a main factor for N- and C-terminal arm becoming ordered upon DNA binding.     

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.     

Drosophila engrailed-1,10-phenanthroline chimeras as probes of homeodomain-DNA complexes.

We have converted the Drosophila engrailed homeodomain into a sequence-specific nuclease by linking the protein to the chemical nuclease 1,10-phenanthroline-copper (OP-Cu). Unique cysteines were introduced at six positions into the homeodomain by site-directed mutagenesis for the covalent attachment of OP-Cu. The varied DNA-binding affinity and specificity of these mutants and the DNA cleavage pattern of their OP-Cu derivatives allowed us to assess the crystal structure of the engrailed homeodomain-DNA complex. We have also achieved site-specific double-stranded DNA scission with one of the homeodomain mutants, E28C, which has the potential of being used to identify engrailed binding sites in the genome. Because the homeodomain is so well conserved among members of the homeodomain-containing protein family, other homeodomain proteins can be converted into nucleases by attaching OP-Cu at position 28 of their homeodomains.     

A phage display selection of engrailed homeodomain mutants and the importance of residue Q50

Mutants of engrailed homeodomain (HD) that retain DNA-binding activity were isolated using a phage display selection. This selection was used to enrich for active DNA-binding clones from a complex library consisting of over a billion members. A more focused library of mutant homeodomains consisting of all possible amino acid combinations at two DNA-contacting residues (I47 and Q50) was constructed and screened for members capable of binding tightly and specifically to the engrailed consensus sequence, TAATTA. The isolated mutants largely recapitulated the distribution of amino acids found at these positions in natural homeodomains thus validating the in vitro selection conditions. In particular, the unequivocal advantage enjoyed by glutamine at residue 50 is surprising in light of reports that minimize the importance of this residue. Here, the subtle contributions of residue Q50 are demonstrated to play a functionally important role in specific recognition of DNA. These results highlight the complex subtlety of protein–DNA interactions, underscoring the value of the first reported in vitro selection of a homeodomain.     

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.     

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.     

Amino acid substitution at the substrate-binding subsite alters the specificity of the Phanerochaete chrysosporium cellobiose dehydrogenase

The active site of cellobiose dehydrogenase from Phanerochaete chrysosporium is composed of two subsites, a catalytic C subsite and a substrate-binding B subsite. Based on the crystal structure of the enzyme with a cellobiose analogue, residue Glu279 was selected for site-directed mutagenesis studies. Substitution of Glu279 to Ala, Asn, and Asp had no effect on the expression of the protein in Pichia pastoris but completely abolished its enzymatic activity. Substitution of Glu279 to Gln drastically altered the enzyme’s substrate specificity. While the wild-type cellobiose dehydrogenase efficiently oxidizes cellobiose and lactose, the Glu279Gln mutant retained most of its activity with cellobiose but was completely inactive with lactose. We generated structural models of the active site interacting with cellobiose and lactose to provide an interpretation of these results.     

On the role of the cis-proline residue in the active site of DsbA

In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis-proline in proximity of the active site residues. In the variant DsbA(P151A), this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild-type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbA(P151A) crystallized in a different crystal form from the wild-type protein, in space group P2(1) with six molecules in the asymmetric unit. Its X-ray structure was determined to 2.8 A resolution. The most significant conformational changes occurred at the active site. The loop 149-152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the asymmetric unit a gauche conformation not observed in the wild-type protein. The X-ray structure and folding studies on DsbA(P151A) were consistent with the cis-proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.     

Altering the DNA-binding specificity of the yeast Matalpha 2 homeodomain protein.

Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism. The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins. To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1. We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein. We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein. Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain. Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.     

Characterization of Cre-loxP interaction in the major groove: hint for structural distortion of mutant Cre and possible strategy for HIV-1 therapy

Although the crystal structure of Cre recombinase complexed with DNA, named loxA, was elucidated a couple of years ago, it has not yet been determined which amino acids of the protein are involved in the specific Cre-loxP interaction. Arg259 and Gln90 interact with DNA substrate in the major groove from which the specificity of protein-DNA interaction comes. In this study, we substituted these residues for other amino acids. Also, two mutated DNA substrates were constructed. In each mutant, one of the bases that interact with Arg259 or Gln90 was changed into another base. In vitro binding assays and recombination assays of variant lox sites with wild-type and mutant-type Cre revealed that Arg259 plays a key role in Cre-loxP binding but Gln90 does not. However, the recombination activity still remained intact, although the binding between Cre and DNA substrate was not ensured.     

Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency

Human angiogenin (Ang) is an RNase in the pancreatic RNase superfamily that induces angiogenesis. Its catalytic activity is comparatively weak, but nonetheless critical for biological activity. The crystal structure of Ang has shown that enzymatic potency is attenuated in part by the obstructive positioning of Gln117 within the B(1) pyrimidine binding pocket, and that the C-terminal segment of residues 117-123 must reorient for Ang to bind and cleave RNA. The native closed conformation appears to be stabilized by Gln117-Thr44 and Asp116-Ser118 hydrogen bonds, as well as hydrophobic packing of Ile119 and Phe120. Consistent with this view, Q117G, D116H, and I119A/F120A variants are 4-30-fold more active than Ang. Here we have determined crystal structures for these variants to examine the structural basis for the activity increases. In all three cases, the C-terminal segment remains obstructive, demonstrating that none of the residues that has been replaced is essential for maintaining the closed conformation. The Q117G structure shows no changes other than the loss of the side chain of residue 117, whereas those of D116H and I119A/F120A reveal C-terminal perturbations beyond the replacement site, suggesting that the native closed conformation has been destabilized. Thus, the interactions of Gln117 seem to be less important than those of residues 116, 119, and 120 for stabilization. In D116H, His116 does not replicate either of the hydrogen bonds of Asp116 with Ser118 and instead forms a water-mediated interaction with catalytic residue His114; residues 117-121 deviate significantly from their positions in Ang. In I119A/F120A, the segment of residues 117-123 has become highly mobile and all of the interactions thought to position Gln117 have been weakened or lost; the space occupied by Phe120 in Ang is partially filled by Arg101, which has moved several angstroms. A crystal structure was also determined for the deletion mutant des(121-123), which has 10-fold reduced activity toward large substrates. The structure is consistent with the earlier proposal that residues 121-123 form part of a peripheral substrate binding subsite, but also raises the possibility that changes in the position of another residue, Lys82, might be responsible for the decreased activity of this variant.     

Full-sequence computational design and solution structure of a thermostable protein variant

Computational protein design procedures were applied to the redesign of the entire sequence of a 51 amino acid residue protein, Drosophila melanogaster engrailed homeodomain. Various sequence optimization algorithms were compared and two resulting designed sequences were experimentally evaluated. The two sequences differ by 11 mutations and share 22% and 24% sequence identity with the wild-type protein. Both computationally designed proteins were considerably more stable than the naturally occurring protein, with midpoints of thermal denaturation greater than 99 degrees C. The solution structure was determined for one of the two sequences using multidimensional heteronuclear NMR spectroscopy, and the structure was found to closely match the original design template scaffold.     

Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7

A conserved cis proline residue located in the active site of Thermotoga maritima acetyl esterase (TmAcE) from the carbohydrate esterase family 7 (CE7) has been substituted by alanine. The residue was known to play a crucial role in determining the catalytic properties of the enzyme. To elucidate the structural role of the residue, the crystal structure of the Pro228Ala variant (TmAcE_P228A) was determined at 2.1 Å resolution. The replacement does not affect the overall secondary, tertiary, and quaternary structures and moderately decreases the thermal stability. However, the wild type cis conformation of the 227–228 peptide bond adopts a trans conformation in the variant. Other conformational changes in the tertiary structure are restricted to residues 222–226, preceding this peptide bond and are located away from the active site. Overall, the results suggest that the conserved proline residue is responsible for the cis conformation of the peptide and shapes the geometry of the active site. Elimination of the pyrrolidine ring results in the loss of van der Waals and hydrophobic interactions with both the alcohol and acyl moeities of the ester substrate, leading to significant impairment of the activity and perturbation of substrate specificity. Furthermore, a cis‐to‐trans conformational change arising out of residue changes at this position may be associated with the evolution of divergent activity, specificity, and stability properties of members constituting the CE7 family. Proteins 2017; 85:694–708. © 2016 Wiley Periodicals, Inc.     

Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 Å) and biochemical basis

Change in specificity, caused by the mutations at P1 site, of the serine protease inhibitors of different families is reported in the literature, but Kunitz (STI) family inhibitors are almost unexplored in this regard. In this paper, we present the crystal structure of a P1 variant of winged bean chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by biochemical, phylogenetic and docking studies on the mutant. A single mutation (Leu → Arg) at P1 converted WCI to a strong inhibitor of trypsin with an association constant of 4.8 × 10¹⁰ M^?1 which is comparable to other potent trypsin inhibitors of the family. The crystal structure (2.15 Å) of this mutant (L65R) shows that its reactive site loop conformation deviates from that of WCI and adopts a structure similar to that of Erythrina caffra trypsin inhibitor (ETI) belonging to the same family. Mutation induced structural changes have also been propagated in a concerted manner to the neighboring conserved scaffolding residue Asn14, such that the side chain of this residue took an orientation similar to that of ETI and optimized the hydrogen bonds with the loop residues. While docking studies provide information about the accommodation of non-specific residues in the active site groove of trypsin, the basis of the directional alteration of the reactive site loop conformation has been understood through sequence analysis and related phylogenetic studies.     

Constitutive regulatory activity of an evolutionarily excluded riboswitch variant

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.     

Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.     

Dioxane contributes to the altered conformation and oligomerization state of a designed engrailed homeodomain variant

Our goal was to compute a stable, full-sequence design of the Drosophila melanogaster engrailed homeodomain. Thermal and chemical denaturation data indicated the design was significantly more stable than was the wild-type protein. The data were also nearly identical to those for a similar, later full-sequence design, which was shown by NMR to adopt the homeodomain fold: a three-helix, globular monomer. However, a 1.65 A crystal structure of the design described here turned out to be of a completely different fold: a four-helix, rodlike tetramer. The crystallization conditions included approximately 25% dioxane, and subsequent experiments by circular dichroism and sedimentation velocity analytical ultracentrifugation indicated that dioxane increases the helicity and oligomerization state of the designed protein. We attribute at least part of the discrepancy between the target fold and the crystal structure to the presence of a high concentration of dioxane.     

Insights into Lou Gehrig's disease from the structure and instability of the A4V mutant of human Cu,Zn superoxide dismutase

We have engineered enhanced DNA-binding function into the a1 homeodomain by making changes in a loop distant from the DNA-binding surface. Comparison of the free and bound a1 structures suggested a mechanism linking van der Waals stacking changes in this loop to the ordering of a final turn in the DNA-binding helix of a1. Inspection of the protein sequence revealed striking differences in amino acid identity at positions 24 and 25 compared to related homeodomain proteins. These positions lie in the loop connecting helix-1 and helix-2, which is involved in heterodimerization with the alpha 2 protein. A series of single and double amino acid substitutions (a1-Q24R, a1-S25Y, a1-S25F and a1-Q24R/S25Y) were engineered, expressed and purified for biochemical and biophysical study. Calorimetric measurements and HSQC NMR spectra confirm that the engineered variants are folded and are equally or more stable than the wild-type a1 homeodomain. NMR analysis of a1-Q24R/S25Y demonstrates that the DNA recognition helix (helix-3) is extended by at least one turn as a result of the changes in the loop connecting helix-1 and helix-2. As shown by EMSA, the engineered variants bind DNA with enhanced affinity (16-fold) in the absence of the alpha 2 cofactor and the variant alpha 2/a1 heterodimers bind cognate DNA with specificity and affinity reflective of the enhanced a1 binding affinity. Importantly, in vivo assays demonstrate that the a1-Q24R/S25Y protein binds with fivefold greater affinity than wild-type a1 and is able to partially suppress defects in repression by alpha 2 mutants. As a result of these studies, we show how subtle differences in residues at a surface distant from the functional site code for a conformational switch that allows the a1 homeodomain to become active in DNA binding in association with its cofactor alpha 2.     

Mutational analysis of the engrailed homeodomain recognition helix by phage display.

The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfamily of eukaryotic gene regulatory proteins. Here, the DNA binding of recognition helix variants of the HD from the engrailed gene of Drosophila melanogaster was investigated by phage display. Nineteen different combinations of pairwise mutations at positions 50 and 54 were screened against a panel of four DNA sequences consisting of the engrailed consensus, a non-specific DNA control based on the lambda repressor operator OR1 and two model sequence targets con-taining imperfect versions of the 5'-TAAT-3' consensus. The resulting mutant proteins could be divided into four groups that varied with respect to their affinity for DNA and specificity for the engrailed consensus. The altered specificity phenotypes of several mutant proteins were confirmed by DNA mobility shift analysis. Lys50/Ala54 was the only mutant protein that exhibited preferential binding to a sequence other than the engrailed consensus. Arginine was also demonstrated to be a functional replacement for Ala54. The functional combinations at 50 and 54 identified by these experiments recapitulate the distribution of naturally occurring HD sequences and illustrate how the engrailed HD can be used as a framework to explore covariation among DNA binding residues.