Methionine-containing zipper peptides

Summary Using circular dichroism, we have examined the effect of single and multiple methionine mutations on the dimerization function of a previously reported engineered leucine zipper peptide. Our results show that the methionine-containing zipper peptides self-associate to form coiled coils that are less stable than that of the reference leucine zipper. The circular dichroism data also indicate that leucine at positiond is more tolerant of methionine substitution than isoleucine at positiona.     

Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: a comparison to Homo sapiens B-ZIP motifs

Basic region-leucine zipper (B-ZIP) proteins are a class of dimeric sequence-specific DNA-binding proteins unique to eukaryotes. We have identified 67 B-ZIP proteins in the Arabidopsis thaliana genome. No A.thaliana B-ZIP domains are homologous with any Homo sapiens B-ZIP domains. Here, we predict the dimerization specificity properties of the 67 B-ZIP proteins in the A.thaliana genome based on three structural properties of the dimeric alpha-helical leucine zipper coiled coil structure: (i) length of the leucine zipper, (ii) placement of asparagine or a charged amino acid in the hydrophobic interface and (iii) presence of interhelical electrostatic interactions. Many A.thaliana B-ZIP leucine zippers are predicted to be eight or more heptads in length, in contrast to the four or five heptads typically found in H.sapiens, a prediction experimentally verified by circular dichroism analysis. Asparagine in the a position of the coiled coil is typically observed in the second heptad in H.sapiens. In A.thaliana, asparagine is abundant in the a position of both the second and fifth heptads. The particular placement of asparagine in the a position helps define 14 families of homodimerizing B-ZIP proteins in A.thaliana, in contrast to the six families found in H.sapiens. The repulsive interhelical electrostatic interactions that are used to specify heterodimerizing B-ZIP proteins in H.sapiens are not present in A.thaliana. Instead, we predict that plant leucine zippers rely on charged amino acids in the a position to drive heterodimerization. It appears that A.thaliana define many families of homodimerizing B-ZIP proteins by having long leucine zippers with asparagine judiciously placed in the a position of different heptads.     

Unfolding of a leucine zipper is not a simple two-state transition

Temperature-induced unfolding of the leucine zipper, an alpha-helical, double-stranded, coiled-coil, was studied by circular dichroism spectroscopy, spectrofluorimetry and heat capacity scanning calorimetry. It is shown that this process does not represent a simple two-state transition, as previously believed, but consists of several stages. The first transition starts at the very beginning of heating from 0 degrees C and proceeds with significant heat absorption and decrease of ellipticity. This transition does not depend on the concentration of protein and is sensitive to modification of the N terminus; it is therefore associated with unfolding or fraying of this part of the leucine zipper. The second transition takes place at a considerably higher temperature; it is more pronounced than the first one and does not depend on the concentration of protein, i.e. it is unimolecular. This transition is sensitive to modification of both termini of the leucine zipper and affects the optical properties of a tryptophan residue placed in the central part of the zipper. It therefore involves the whole dimer but does not result in its dissociation, presumably being associated with some repacking of the coiled-coil. This second transition is followed at higher temperatures by the concentration-dependent cooperative unfolding/dissociation of the two strands. The enthalpy and entropy of the temperature-induced structural changes of the leucine zipper that take place before its cooperative unfolding/dissociation comprises almost 40% of the total enthalpy and entropy of unfolding of the completely folded coiled-coil, the state in which it appears to be below 0 degrees C. Comparison of the total enthalpy of leucine zipper unfolding with that of a single-stranded alpha-helix shows that their temperature-dependence correlates with the extent of intramolecular non-polar contacts and allows an assessment of the enthalpy of hydrogen bonding in alpha-helices, which appears to be about 3.3kJmol(-1) at 20 degrees C.     

Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position.

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (delta delta GP = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol-1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (delta delta GP = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by delta delta GP = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.     

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1,3)-Ac2-alpha-D-GalN3+ ++]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CalphaH chemical shift perturbations, 3JNH:CalphaH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.     

Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and confirmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial alpha-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.     

Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of B-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS. We describe a dominant negative protein, A-VBP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VBP basic region in the VBP.A-VBP heterodimer. This new alpha-helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol. A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND. Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <--> e' or i, i' +5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBPalpha leucine zipper to contain the g <--> e' salt bridges that characterize VBP. A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA. These conserved g <--> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.     

Dissociation and unfolding of GCN4 leucine zipper in the presence of sodium dodecyl sulfate.

The dissociation and unfolding behavior of the GCN4 leucine zipper has been studied using SDS titration. Circular dichroism (CD) spectra showed that the alpha-helix content of the leucine zipper (20 microM) decreased during the sodium dodecyl sulfate (SDS) titration. However, the alpha-helix content of the leucine zipper still remained significant in the presence of 1 mM SDS, with little change detected when the SDS concentration further increased to 2 mM. The dimer dissociation of the leucine zipper is also a co-operative process during SDS titration; with no dimer remaining when SDS concentration reached 1 mM, as shown by electrophoresis and the the theta(222)/theta(208) ratio. Our results indicate that SDS efficiently induces leucine zipper dimer dissociation with the monomers still partially folded. The experimental results provide important evidence for the previous model that partial helix formation precedes dimerization in coiled coil folding.     

The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor

The human protein NEFA (DNA binding, EF-hand, Acidic region) has previously been isolated from a KM3 cell line and immunolocalized on the plasma membrane, in the cytoplasma, and in the culture medium. Sequence analysis of a cDNA clone encoding NEFA identified a hydrophilic domain, two EF-hands, and a leucine zipper at the C- terminus. These characters are shared with nucleobindin (Nuc). In this paper we have further characterized NEFA and probed its evolutionary origins. Circular dichroism (CD) spectra of recombinant NEFA indicated a helical content of 51% and showed that the EF-hands are capable of binding Ca2+. Experiments with recombinant NEFA and synthesized peptides revealed that the leucine zipper cannot form a homodimer. The leucine zipper may allow heterodimer formation of NEFA and an unknown protein. Phylogenetic analyses suggest that this protein is derived from a four-domain EF-hand ancestor with subsequent duplications and fusions. The leucine zipper and putative DNA-binding domains of NEFA have evolved secondarily from existing EF-hand sequences. These analyses provide insights into how complex proteins may originate and trace the precursor of NEFA to the common ancestor of eukaryotes.      

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).     

An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway

The leucine zipper is a dimeric coiled-coil structural motif consisting of four to six heptad repeats, designated (abcdefg)(n). In the GCN4 leucine zipper, a position 16 in the third heptad is occupied by an Asn residue whereas the other a positions are Val residues. Recently, we have constructed variants of the GCN4 leucine zipper in which the a position Val residues were replaced by Ile. The folding and unfolding of the wild-type GCN4 leucine zipper and the Val to Ile variant both adhere to a simple two-state mechanism. In this study, another variant of the GCN4 leucine zipper was constructed by moving the single Asn residue from a position 16 to a position 9. This switch causes the thermal unfolding of the GCN4 leucine zipper to become three state. The unfolding pathway of this variant was determined by thermal denaturation, limited proteinase K digestion, and sedimentation equilibrium analysis. Our data are consistent with a model in which the variant first unfolds from its N terminus and changes the oligomerization specificity from a native dimer to a partially unfolded intermediate containing a mixture of dimers and trimers and then completely unfolds to unstructured monomers.     

The roles of side chain and backbone in protein structure probed with glycine- and sarcosine-rich synthetic leucine zipper peptides

The protein folding problem has long been a formidable challenge. Here we present a synthetic natural motif approach that exploits small preexisting structural models for the dissection of forces important in protein folding. An example for this approach is shown in the modification of a 31-residue leucine zipper peptide with the helix-breaking amino acid glycine and the hydrogen bond-breaking imino acid sarcosine. Circular dichroism and NMR experiments have shown that the glycine-modified leucine zipper peptide adopts a stable helical conformation similar to the native conformation while the sarcosine-modified leucine zipper peptide adopts a random coil conformation. These results provide valuable insight into the current controversy over the relative importance of long-range side chain-side chain interactions versus local backbone interactions in protein structure and suggest that the natural motif strategy may represent a useful model to study protein folding.     

Engineering of an environmentally responsive beta roll peptide for use as a calcium-dependent cross-linking domain for peptide hydrogel formation

We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development.     

The DNA-binding domain of the Cys-3 regulatory protein of Neurospora crassa is bipartite.

Cys-3, the major sulfur regulatory gene of Neurospora crassa, encodes a regulatory protein that is capable of sequence-specific interaction with DNA. The interaction is mediated by a region within the CYS3 protein (the bzip region) which contains a potential dimer-forming surface, the leucine zipper, and an adjacent basic DNA contact region, NH2-terminal to the leucine zipper. To investigate the bipartite nature of the bzip region, a series of cys-3 mutants obtained by oligonucleotide-directed mutagenesis were expressed and tested for dimer formation as well as DNA binding and in vivo function. The results demonstrate that CYS3 protein exists as a dimer in the presence and absence of the target DNA and that dimerization of CYS3 is mediated strictly by the leucine zipper, which is required for both cys-3 function in vivo and DNA-binding activity in vitro. Furthermore, a truncated CYS3 protein corresponding to just the bzip region was found to mediate dimer formation and to possess DNA-binding activity. A CYS3 mutant protein with a pure methionine zipper showed significant, although reduced, function in vivo and in vitro.