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.     

Determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution by 1H nuclear magnetic resonance spectroscopy

The determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution is described. The techniques used are 1H nuclear magnetic resonance spectroscopy for the data collection, and calculation of the protein structure with the program DISMAN followed by restrained energy minimization with a modified version of the program AMBER. A group of 19 conformers characterizes a well-defined structure for residues 7 to 59, with an average root-mean-square distance from the backbone atoms of 0.6 A relative to the mean of the 19 structures. The structure contains a helix from residues 10 to 21, a helix-turn-helix motif from residues 28 to 52, which is similar to those reported for several prokaryotic repressor proteins, and a somewhat flexible fourth helix from residues 53 to 59, which essentially forms an extension of the presumed recognition helix, residues 42 to 52. The helices enclose a structurally well-defined molecular core of hydrophobic amino acid side-chains.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain.

The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well-known helix-turn-helix motif. The recognition helix penetrates the DNA major groove, gives specific protein-DNA contacts and forms direct, or water-mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42-53, where residues W48 and F49, not involved in the protein-DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well-defined secondary structure in TFE and in TFE/water (90/10, v/v) with an alpha-helix encompassing residues 4-9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted alpha-helical conformation involving residues 4-8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the alpha-helical conformation even if separated from the rest of the molecule.     

Comparison of penetratin and other homeodomain-derived cell-penetrating peptides: interaction in a membrane-mimicking environment and cellular uptake efficiency

Antennapedia and other homeoproteins have the unique ability to efficiently translocate across biological membranes, a property that is mediated by the third helix of the homeodomain. To analyze the effects of sequence divergence in the homeodomain, we have compared the cellular uptake efficiencies and interaction properties in a membrane-mimicking environment of four peptides corresponding to the third helix sequence of Antennapedia, Engrailed-2, HoxA-13, and Knotted-1. NMR studies revealed that these peptides adopt helical conformations in SDS micelles. Their localization with respect to the micelle was investigated using Mn(2+) as a paramagnetic probe. Peptides are positioned parallel to the micelle surface, but subtle differences in the depth of immersion were observed. Using a recently developed method for quantification of CPP cellular uptake based on MALDI-TOF mass spectrometry, all of these peptides were found to translocate into cells but with large differences in their uptake efficiencies. The peptide with the highest uptake efficiency was found to be the least deeply inserted within the micelle, indicating that electrostatic surface interactions may be a major determinant for membrane translocation. A new cell-penetrating peptide derived from Knotted-1 homeodomain with improved uptake properties compared to penetratin is introduced here.     

Efficient Intracellular Delivery of GFP by Homeodomains of Drosophila Fushi-tarazu and Engrailed Proteins

The 60 amino acid long homeodomain of Antennapedia (Antp), either alone or as a fusion protein with 30-40 amino acid long foreign polypeptides, has been reported to cross biological membranes by an energy- and receptor-protein-independent mechanism. Moreover, the 16 amino acid long third helix of the Antp homeodomain, so-called penetratin, possesses translocation properties when fused to fewer than 100 amino acids as well. These findings led us to study whether such a protein tansduction property is shared by other homeodomains. We report here that homeodomains of two homeoproteins, Fushi-tarazu and Engrailed, are able to transduce a 238 amino acid long green fluorescent protein into cultured cells as efficiently as other well-known protein transduction domains, such as an internal oligopeptide of Tat and penetratin. These findings suggest that such transduction activity of homeodomains might have some physiological roles and that it can be exploited for development of efficient transduction vectors for research use and protein therapy.     

Translocation of the pAntp peptide and its amphipathic analogue AP-2AL

The pAntp peptide, corresponding to the third helix of the homeodomain of the Antennapedia protein, enters by a receptor-independent process into eukaryotic cells. The interaction between the pAntp peptide and the phospholipid matrix of the plasma membrane seems to be the first step involved in the translocation mechanism. However, the mechanism by which the peptide translocates through the cell membrane is still not well established. We have investigated the translocation ability of pAntp through a protein-free phospholipid membrane in comparison with a more amphipathic analogue. We show by fluorescence spectroscopy, circular dichroism, NMR spectroscopy, and molecular modeling that pAntp is not sufficiently helically amphipathic to cross a phospholipid membrane of a model system. Due to its primary sequence related to its DNA binding ability in the Antennapedia homeodomain-DNA complex, the pAntp peptide does not belong to the amphipathic alpha-helical peptide family whose members are able to translocate by pore formation.     

Cellular uptake of cationic gadolinium-DOTA peptide conjugates with and without N-terminal myristoylation

Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373 glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20% of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake.     

Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin.

Peptides derived from the third alpha-helix of the homeodomain (residues 43-58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N- and C-terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C-terminal segment 52Arg-Arg-Met-Lys-Trp-Lys-Lys58 of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide's basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.     

Efficient Intracellular Delivery of GFP by Homeodomains of Drosophila Fushi-tarazu and Engrailed Proteins

The 60 amino acid long homeodomain of Antennapedia (Antp), either alone or as a fusion protein with 30–40 amino acid long foreign polypeptides, has been reported to cross biological membranes by an energy- and receptor-protein-independent mechanism. Moreover, the 16 amino acid long third helix of the Antp homeodomain, so-called penetratin, possesses translocation properties when fused to fewer than 100 amino acids as well. These findings led us to study whether such a protein tansduction property is shared by other homeodomains. We report here that homeodomains of two homeoproteins, Fushi-tarazu and Engrailed, are able to transduce a 238 amino acid long green fluorescent protein into cultured cells as efficiently as other well-known protein transduction domains, such as an internal oligopeptide of Tat and penetratin. These findings suggest that such transduction activity of homeodomains might have some physiological roles and that it can be exploited for development of efficient transduction vectors for research use and protein therapy.     

A single amino acid can determine the DNA binding specificity of homeodomain proteins

Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.     

Positively charged residues at the N-terminal arm of the homeodomain are required for efficient DNA binding by homeodomain-leucine zipper proteins

Plant homeodomain-leucine zipper proteins, unlike most animal homeodomains, bind DNA efficiently only as dimers. In the present work, we report that the deletion of the homeodomain N-terminal arm (first nine residues) of the homeodomain-leucine zipper protein Hahb-4 dramatically affects its DNA-binding affinity, causing a 70-fold increase in dissociation constant. The addition of the N-terminal arm of Drosophila Antennapedia to the truncated form restores the DNA-binding affinity of dimers to values similar to those of the native form. However, the Antennapedia N-terminal arm is not able to confer increased binding affinity to monomers of Hahb-4 lacking the leucine zipper motif, indicating that the inefficient binding of monomers must be due to structural differences in other parts of the molecule. The construction of proteins with modifications at residues 5 to 7 of the homeodomain suggests strongly that positively charged amino acids at these positions play essential roles in determining the DNA-binding affinity. However, the effect of mutations at positions 6 and 7 can be counteracted by introducing a stretch of positively charged residues at positions 1 to 3 of the homeodomain. Sequence comparisons indicate that all homeodomain-leucine zipper proteins might use contacts of the N-terminal arm with DNA for efficient binding. The occurrence of a homeodomain with a DNA-interacting N-terminal arm must then be an ancient acquisition in evolution, earlier than the separation of lines leading to metazoa, fungi and plants.     

Cooperative DNA‐binding and sequence‐recognition mechanism of aristaless and clawless

To achieve accurate gene regulation, some homeodomain proteins bind cooperatively to DNA to increase those site specificities. We report a ternary complex structure containing two homeodomain proteins, aristaless (Al) and clawless (Cll), bound to DNA. Our results show that the extended conserved sequences of the Cll homeodomain are indispensable to cooperative DNA binding. In the Al–Cll–DNA complex structure, the residues in the extended regions are used not only for the intermolecular contacts between the two homeodomain proteins but also for the sequence‐recognition mechanism of DNA by direct interactions. The residues in the extended N‐terminal arm lie within the minor groove of DNA to form direct interactions with bases, whereas the extended conserved region of the C‐terminus of the homeodomain interacts with Al to stabilize and localize the third α helix of the Cll homeodomain. This structure suggests a novel mode for the cooperativity of homeodomain proteins.     

Interaction of fusogenic peptides with an antisense oligonucleotide in solution and in the presence of micelles: conformational studies

The conformational effect of the interaction between various fusogenic peptides and an 18mer single stranded antisense oligonucleotide (ODN), targeted towards the green fluorescent protein mRNA, has been studied by circular dichroism spectroscopy in water and in the presence of anionic lysolipid micelles. The peptides used were the third helix of Antennapedia homeodomain pAntp-(43-58), the flock house virus FHV-gamma-(364-407) peptide, and its N-terminal gamma1-(364-384) and C-terminal gamma2-(390-407) fragments. The most significant conformational changes were observed in ODN-pAntp-(43-58) and ODN-FHV-gamma2 complexes. The pAntp-(43-58) forms a complex with ODN through electrostatic interaction resulting in profound changes in the conformation of both the peptide and the ODN. In the case of FHV-gamma2 peptide the complex formation takes place without altering the structure of ODN, and the decreased ratio of deltaepsilon208/deltaepsilon222 reflects the insertion of the complexed peptide into the micelle.     

Antennapedia/HS1 chimeric phosphotyrosyl peptide: conformational properties, binding capability to c-Fgr SH2 domain and cell permeability.

With the aim of interfering with the signaling pathways mediated by the SH2 domains of Src-like tyrosine kinases, we synthesized a tyrosyl-phospho decapeptide, corresponding to the sequence 392-401 of HS1 protein, which inhibits the secondary phosphorylation of HS1 protein catalyzed by the Src-like kinases c-Fgr or Lyn. This phospho-peptide was modified to enter cells by coupling to the third helix of Antennapedia homeodomain, which is able to translocate across cell membranes. Here we present CD and fluorescence studies on the conformational behavior in membrane-mimicking environments and on lipid interactions of Antennapedia fragment and its chimeric phosphorylated and unphosphorylated derivatives. These studies evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption on lipids. Experiments performed with recombinant protein containing the SH2 domain of c-Fgr fused with GST and with isolated erythrocyte membranes demonstrated that the presence of the N-terminal Antennapedia fragment only slightly affects the binding of the phospho-HS1 peptide to the SH2 domain. In fact, it has been shown that in isolated erythrocyte membranes, both phospho-HS1 peptide and its chimeric derivative greatly affect either the SH2-mediated recruitment of the c-Fgr to the transmembrane protein band 3 and the following phosphorylation of the protein catalyzed by the Src-like kinase c-Fgr. The ability of the chimeric phospho-peptide to enter cells has been demonstrated by confocal microscopy analysis.     

Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR.

The Antennapedia homeodomain protein of Drosophila has the ability to penetrate biological membranes and the third helix of this protein, residues 43-58, known as penetratin (RQIKIWFQNRRMKWKK-amide) has the same translocating properties as the entire protein. The variant, RQI KIFFQNRRMKFKK-amide, here called penetratin (W48F,W56F) does not have the same ability. We have determined a solution structure of penetratin and investigated the position of both peptides in negatively charged bicelles. A helical structure is seen for residues Lys46 through Met54. The secondary structure of the variant penetratin(W48F,W56F) in bicelles appears to be very similar. Paramagnetic spin-label studies and analysis of NOEs between penetratin and the phospholipids show that penetratin is located within the bicelle surface. Penetratin (W48F,W56F) is also located inside the phospholipid bicelle, however, with its N-terminus more deeply inserted than that of wild-type penetratin. The subtle differences in the way the two peptides interact with a membrane in an equilibrium situation could be important for their translocating ability. As a comparison we have also investigated the secondary structure of penetratin(W48F,W56F) in SDS micelles and the results show that the structure is very similar in SDS and bicelles. In contrast, penetratin(W48F,W56F) and penetratin appear to be located differently in SDS micelles. This clearly shows the importance of using realistic membrane mimetics for investigating peptide-membrane interactions.     

Conserved classes of homeodomains in Schistosoma mansoni, an early bilateral metazoan.

Genes containing a homeobox can be divided into classes based on the distinctive peptide sequences of their diverged homeodomains. Many of these classes, including Antennapedia, engrailed and paired, are strongly conserved in higher multicellular animals, but have not previously been found in platyhelminths, the flatworms which represent the most primitive bilateral metazoans. We have screened cDNA libraries of the platyhelminth Schistosoma mansoni using a degenerate oligonucleotide derived from the third helix of the homeodomain, and have identified numerous schistosome homeobox-containing sequences, including members of the Antennapedia, engrailed and paired classes. The schistosome homeodomain sequences are more similar to the higher animals sequences in their respective classes than they are to each other, indicating that the establishment of these three distinctive classes is at least as ancient as the flatworms. Our data suggest that the ancestral functions of the Antennapedia, engrailed and paired classes involve fundamental features of all bilateral metazoan development. The putative full-length coding sequence of the S. mansoni en homologue is presented.     

Homeoboxes in flatworms.

A search for homeobox-containing genes was done in the genome of a primitive metazoan, the parasitic tapeworm Echinococcus granulosus. Five different homeoboxes were isolated, none of them belonging to the classical Antennapedia-type. Three of the homeodomains are similar to those from the Drosophila melanogaster NK-type genes. The fourth homeodomain shares extensive identity with that of the recently reported homeobox-containing gene goosecoid from Xenopus laevis. The third helix (the recognition helix) of the fifth isolated homeodomain is identical to that of the Xlim-1 gene of X. laevis and the lin11 gene of Caenorharbditis elegans. Using PCR, some Antennapedia-type homeoboxes were cloned from the genome of two other Platyhelminthes, Dugesia tigrina (planaria) and Fasciola hepatica. These data suggest that, contrary to what is found for the majority of the more complex metazoans, Platyhelminthes contain few homeobox genes belonging to the Antennapedia-type.