A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions.

The leucine zipper is a dimeric coiled-coil protein structure composed of two amphipathic alpha-helices with the hydrophobic surfaces interacting to create the dimer interface. This structure has been found to mediate the dimerization of two abundant classes of DNA binding proteins: the bZIP and bHLH-Zip proteins. Several workers have reported that amino acids in the e and g positions of the coiled coil can modulate dimerization stability and specificity. Using the bZIP protein VBP as a host molecule, we report a thermodynamic scale (delta delta G) for 27 interhelical interactions in 35 proteins between amino acids in the g and the following e positions (g<==>e') of a leucine zipper coiled coil. We have examined the four commonly occurring amino acids in the e and g positions of bZIP proteins, lysine (K), arginine (R), glutamine (Q), glutamic acid (E), as well as the only other remaining charged amino acid aspartic acid (D), and finally alanine (A) as a reference amino acid. These results indicate that E<==>R is the most stable interhelical pair, being 0.35 kcal/mol more stable than E<==>K. A thermodynamic cycle analysis shows that the E<==>R pair is 1.33 kcal/mol more stable than A<==>A with -1.14 kcal/mol of coupling energy (delta delta Gint) coming from the interaction of E with R. The E<==>K coupling energy is only -0.14 kcal/mol. E interacts with more specificity than Q. The R<==>R pair is less stable than the K<==>K by 0.24 kcal/mol. R interacts with more specificity than K. Q forms more stable pairs with the basic amino acids K and R rather than with E. Changing amino acids in the e position to A creates bZIP proteins that form tetramers.     

Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.     

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.     

Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at ‘a’ and/or ‘d’ position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its ‘a’ and ‘d’ positions with d-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its d-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at ‘a’ and/or ‘d’ positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.     

The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein.

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of molecular weight 78,000 (gp78), mediating cell motility signaling in vitro and metastasis in vivo. Here, we cloned the full-length cDNAs for both human and mouse AMFR genes. Both genes encode a protein of 643 amino acids containing a seven transmembrane domain, a RING-H2 motif and a leucine zipper motif and showed a 94.7% amino acid sequence identity to each other. Analysis of the amino acid sequence of AMFR with protein databases revealed no significant homology with all known seven transmembrane proteins, but a significant structural similarity to a hypothetical protein of Caenorhabditis elegans, F26E4.11. Thus, AMFR is a highly conserved gene which encodes a novel type of seven transmembrane protein.     

Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1

We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met. It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif. The glutathione S-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376–405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380–408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction. The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48. These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein–protein interactions may not be necessary for this interaction of chHAT-1.     

Molecular cloning,expression, overproduction and characterization of human TRAIP Leucine zipper protein

The TRAIP interacting protein is known as a negative regulator of TNF-induced-nuclear factor, kappa-light-chain-enhancer of activated B cell (NF-κB) by direct interaction with the adaptor protein TRAF2, which inhibits the function of TRAF2 via the RINGCC domain protein. The TRAIP protein is composed of 469 amino acids with an N-terminal RING motif that is followed by a coiled coil (CC) and leucine zipper domain. TRAIP proteins are critical in programmed cell death, cell proliferation and differentiation, and embryonic development. The critical functions of TRAIP together with the molecular inhibitory mechanism effect of TRAIP have been reported by two different studies and have opened up new research into the field of TRAF biology. In this study, we designed different constructs of the Leucine zipper domain to find the over –expressed construct for further studies. We successfully cloned the C-terminal TRAIP containing the leucine zipper domain. In addition, we have over-expressed and purified the TRAIP LZ for their biochemical characterization.     

Inducing toxicity by introducing a leucine-zipper-like motif in frog antimicrobial peptide, magainin 2

Cytotoxicity, a major obstacle in therapeutic application of antimicrobial peptides, is controlled by leucine-zipper-like sequences in melittin and other naturally occurring antimicrobial peptides. Magainin 2 shows significantly lower cytotoxicity than many naturally occurring antimicrobial peptides and lacks this structural element. To investigate the consequences of introducing a leucine zipper sequence in magainin 2, a novel analogue (Mag-mut) was designed by rearranging only the positions of its hydrophobic amino acids to include this structural element. Both magainin 2 and Mag-mut showed appreciable similarities in their secondary structures in the presence of negatively charged lipid vesicles, in localizing and permeabilizing the selected bacteria and exhibiting bactericidal activities. However, Mag-mut bound and localized strongly on to the mammalian cells tested and exhibited significantly higher cytotoxicity than magainin 2. Only Mag-mut, but not magainin 2, permeabilized human red blood cells and zwitterionic lipid vesicles. In contrast with magainin 2, Mag-mut self-assembled in an aqueous environment and bound co-operatively on to zwitterionic lipid vesicles. The peptides formed pores of different sizes on to a selected mammalian cell. The results of the present study indicate an important role of the leucine zipper sequence in the cytotoxicity of Mag-mut and demonstrate that its introduction into a non-toxic peptide, without altering the amino acid composition, can render cytotoxicity.     

Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.     

Protein product encoded by a human novel gene E9730 enhances AP-1 activity through interacting with Jab1

A novel human gene, named E9730 (a clone number of fetal liver cDNA library), has been identified from more than 14,000 expressed sequence tags (ESTs) based on our large scale sequencing of human fetal liver cDNA libraries. Although sequencing of this novel human gene indicates that it is a leucine zipper protein, the function of E9730 and its homologous genes among species is unknown yet. To find out physiological functional clue of E9730, the yeast two-hybrid system was used to screen the E9730-interac-ting protein(s), and one clone containing a cDNA insert with almost the entire coding sequence (amino acids 39-335) of human Jabl (Jun-activating domain binding protein 1) that interacted specifically with E9730 was identified. A specific association between Jabl and E9730 was shown by co-immunoprecipitation and co-localization experiments. Furthermore, E9730 appeared to enhance Jab 1 -induced AP-1 activity in a concentration-dependent manner and Jabl may be involved in the intracellular signaling trans     

Molecular cloning of nucleobindin, a novel DNA-binding protein that contains both a signal peptide and a leucine zipper structure.

We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mouse produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55 kD protein that we termed nucleobindin (Nuc), and obtained its cDNA clone. Although the gene for Nuc encodes a signal peptide and, in fact, Nuc was identified as a secreted protein, Nuc had a DNA-binding property. The putative polypeptide predicted from the cDNA sequence featured a signal peptide, a leucine zipper structure and a basic amino acid-rich region. The DNA-binding property of Nuc was destroyed by deletion of either the leucine zipper structure or the basic amino acid-rich region. The amino acid sequences of Nuc are highly conserved between mouse and human. We discuss the possible role of Nuc in autoimmunity.     

Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein.

The paramyxovirus fusion proteins have a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit (R. Buckland and F. Wild, Nature [London] 338:547, 1989). To determine the role of the conserved leucines in the oligomeric structure and biological activity of the Newcastle disease virus (NDV) fusion protein, the heptadic leucines at amino acids 481, 488, and 495 were changed individually and in combination to an alanine residue. While single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein, although cell surface expression of the mutants was higher than that of the wild-type protein. Substitution of all three leucine residues with alanine did not alter the size of the fusion protein oligomer as determined by sedimentation in sucrose gradients. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain, resulted in secretion of an oligomeric polypeptide. These results indicate that the conserved leucines are not necessary for oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative changes of serine to alanine (position 473), glutamic acid to lysine or alanine (position 482), asparagine to lysine (position 485), or aspartic acid to alanine (position 489), the fusogenic ability of the protein was not significantly disrupted. In addition, a double mutant (E482A,D489A) which removed negative charges along one side of the helix had negligible effects on fusion activity.     

A heparin binding protein whose expression increases during differentiation of embryonal carcinoma cells to parietal endoderm cells: cDNA cloning and sequence analysis

A cDNA clone isolated from a lambda gt11 expression library of teratocarcinoma OTT6050 specifies for a glycoprotein with a molecular weight of about 44,000. The new glycoprotein was termed heparin binding protein-44 (HBP-44), since it was absorbed to a heparin-agarose column and was eluted from it by a buffer containing 1.5 M NaCl. HBP-44 mRNA was intensely expressed in PYS-2 parietal endoderm cells and in the kidney, and the RNA level increased about 10-fold during differentiation of F9 embryonal carcinoma cells to parietal endoderm cells. From the cDNA sequence, HBP-44 was concluded to be rich in charged amino acids, and large segments of the protein appeared to form alpha-helixes. The protein was considered to be anchored to the membrane by a cluster of hydrophobic amino acids present in the N-terminal region. Indeed, the N-terminal sequence of HBP-44 was homologous to asialoglycoprotein receptor, which is anchored to the membrane by the N-terminal region. Furthermore, a portion of the N-terminal region of HBP-44 was homologous to the leucine zipper domain. Except for the N-terminal region, HBP-44 had over-all homology with structural proteins such as myosin heavy chain. We propose that HBP-44 is extruded from plasma membranes and interacts with heparin and related molecules and that it is involved in the interactions of plasma membranes with basement membranes.