Evidence for leucine zipper motif in lactose repressor protein

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.     

The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.     

Retrovirus integrase: identification of a potential leucine zipper motif.

The secondary structure of the retrovirus integration protein (IN) was predicted from seven inferred retrovirus IN sequences. The IN sequences were aligned by computer and the phylogenetic relationships between them were determined. The secondary structure of the aligned IN sequences was predicted by two consensus prediction methods. The predicted secondary structural patterns from the two consensus prediction schemes were compared with and superimposed on a composite structural profile of hydropathic/chain flexibility/amphipathic indexes with each index profile being calculated independently for the aligned IN sequences. The use of this composite structural profile not only enhanced the prediction accuracy but also helped in defining the surface loop regions which would be otherwise unpredictable by the use of consensus prediction methods alone. An amphipathic helix was identified by these united structural prediction-chain property profiles. Helical wheel analysis gave the amphipathic helix a coiled-coil like pattern which was similar to the leucine zipper discovered for some eukaryotic gene regulatory proteins. The proposed amphipathic helix may play an essential role in defining the biological properties of IN.     

Cytoplasmic envelopment of human cytomegalovirus requires the postlocalization function of tegument protein pp28 within the assembly compartment

The assembly of herpesvirus remains incompletely defined due to the structural complexity of these viruses. Although the assembly of the capsid of these large DNA viruses is well studied and reasonably well conserved for all members of this diverse family of viruses, the cytoplasmic processes of tegumentation and envelopment are not well understood. The virion of the largest human herpesvirus, human cytomegalovirus (HCMV), contains over 70 virus-encoded proteins that are incorporated during a nuclear and cytoplasmic phase of assembly. Envelopment of this virus requires the function of at least one tegument protein, pp28, the product of the UL99 open reading frame. However, the role of pp28 in the envelopment of HCMV remains undefined. We have generated a pp28 mutant virus that encodes only the first 50 amino acids (aa) of this 190-aa virion protein. This virus is replication impaired and is defective in virus assembly. Characterization of both intracellular and extracellular virions from cells infected with this viral mutant indicated that the decrease in production of infectious virus was secondary to a defect in envelopment and the accumulation of tegumented, noninfectious intracellular particles. Image analysis using fluorescence recovery after photobleaching indicated that the pp28 mutant protein encoded by this virus failed to efficiently accumulate in the virus assembly compartment (AC). Our results suggest that pp28 must accumulate in the AC for efficient envelopment of the particle and provide evidence for a direct role of this tegument protein in the late stages of assembly, such as envelopment.     

Multimerization of tegument protein pp28 within the assembly compartment is required for cytoplasmic envelopment of human cytomegalovirus

Human cytomegalovirus (HCMV) UL99-encoded pp28 is an essential tegument protein required for envelopment and production of infectious virus. Nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with the UL99 gene deleted. Previous results have suggested that a key function of pp28 in the envelopment of infectious HCMV is expressed after the protein localizes in the assembly compartment (AC). In this study, we investigated the potential role of pp28 multimerization in the envelopment of the infectious virion. Our results indicated that pp28 multimerized during viral infection and that interacting domains responsible for self-interaction were localized in the amino terminus of the protein (amino acids [aa] 1 to 43). The results from transient-expression and/or infection assays indicated that the self-interaction took place in the AC. A mutant pp28 molecule containing only the first 35 aa failed to accumulate in the AC, did not interact with pp28 in the AC, and could not support virus replication. In contrast, the first 50 aa of pp28 was sufficient for the self-interaction within the AC and the assembly of infectious virus. Recombinant viruses encoding an in-frame deletion of aa 26 to 33 of pp28 were replication competent, whereas infectious virus was not recovered from HCMV BACs lacking aa 26 to 43. These findings suggested that the accumulation of pp28 was a prerequisite for multimerization of pp28 within the AC and that pp28 multimerization in the AC represented an essential step in the envelopment and production of infectious virions.     

The human cytomegalovirus (HCMV) tegument protein UL94 is essential for secondary envelopment of HCMV virions

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood. However, several tegument proteins are known to be essential for proper particle assembly and maturation. Despite intense investigation, the function of many tegument proteins remains unknown. The HCMV UL94 gene is conserved among all herpesviruses and encodes a virion protein of unknown function. We demonstrate here that UL94 is a tegument protein that is expressed with true-late kinetics and localizes to the viral assembly complex during infection. To elucidate the function of UL94, we constructed a UL94-null mutant, designated UL94stop. This mutant is completely defective for replication, demonstrating that UL94 is essential. Phenotypic analysis of the UL94stop mutant shows that in the absence of UL94, viral gene expression and genome synthesis occur at wild-type levels. However, analysis of the localization of viral proteins to the cytoplasmic assembly complex shows that the essential tegument protein UL99 (pp28) exhibits aberrant localization in cells infected with the UL94stop mutant. Finally, we show that there is a complete block in secondary envelopment in the absence of UL94. Taken together, our data suggest that UL94 functions late in infection to direct UL99 to the assembly complex, thereby facilitating secondary envelopment of virions.     

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 novel streptococcal leucine zipper protein (Lzp) binds to human immunoglobulins

Streptococcus pyogenes is an important pathogen that causes pharyngitis, scarlet fever, rheumatic fever, and streptococcal toxic shock syndrome. To survive within its host, S. pyogenes has developed several immune evasion mechanisms. Here, we identified a novel gene encoding a 66-kDa protein with many leucine zipper motifs, that we call streptococcal leucine zipper protein (Lzp). Lzp was expressed on the bacterial cell surface, and some was detected in the culture medium. Lzp was expressed by all the S. pyogenes strains we tested, but not by group B streptococcal strains. Western blotting and Biacore assay demonstrated that recombinant Lzp bound to human IgA, IgG, IgM, and Lzp. In addition, native-PAGE analysis suggested that the Lzp molecule formed dimer and trimer conformations. Thus, Lzp is a novel immunoglobulin-binding protein that may play a role in helping S. pyogenes escape detection by the host immune system.     

IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif

Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA. OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB. This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif. OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro. The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper. We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction. Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function. Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex". Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats.     

A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif.

The homeobox, a 183 bp DNA sequence element, was originally identified as a region of sequence similarity between many Drosophila homeotic genes. The homeobox codes for a DNA-binding motif known as the homeodomain. Homeobox genes have been found in many animal species, including sea urchins, nematodes, frogs, mice and humans. To isolate homeobox-containing sequences from the plant Arabidopsis thaliana, a cDNA library was screened with a highly degenerate oligonucleotide corresponding to a conserved eight amino acid sequence from the helix-3 region of the homeodomain. Using this strategy two cDNA clones sharing homeobox-related sequences were identified. Interestingly, both of the cDNAs also contain a second element that potentially codes for a leucine zipper motif which is located immediately 3'' to the homeobox. The close proximity of these two domains suggests that the homeodomain-leucine zipper motif could, via dimerization of the leucine zippers, recognize dyad-symmetrical DNA sequences.     

Molecular dynamics simulation of a leucine zipper motif predicted for the integrase of human immunodeficiency virus type I

We have used the molecular dynamics (MD) simulation package AMBER4 to search the conformation of a peptide predicted as a leucine zipper motif for the human immunodeficiency virus type I integrase protein (HIV IN-LZM). The peptide is composed of 22 amino acid residues and its location is from Val 151 to Leu 172. The searching procedure also includes two known α-helices that served as positive controls—namely, a 22-residue GCN4-p1 (LZM) and a 20-residue poly(L -alanine) (PLA). A 21-residue peptide extracted from a cytochrome C crystal (CCC-t) with determined conformation as a β-turn is also included as a negative control. At the beginning of the search, two starting conformations—namely, the standard right-handed α-helix and the fully stretched conformations—are generated for each peptide. Structures generated as standard α-helix are equilibrated at room temperature for 90 ps while structures generated as a fully stretched one are equilibrated at 600 K for 120 ps. The CCC-t and PLA helices are nearly destroyed from the beginning of equilibration. However, for both the HIV IN-LZM and the GCN4-p1 LZM structures, there is substantial helicity being retained throughout the entire course of equilibration. Although helix propagation profiles calculated indicate that both peptides possess about the same propensity to form an α-helix, the HIV IN-LZM helix appears to be more stable than the GCN4-p1 one as judged by a variety of analyses on both structures generated during the equilibration course. The fact that predicted HIV IN-LZM can exist as an α-helix is also supported by the results of high temperature equilibration run on the fully stretched structures generated. In this run, the RMS deviations between the backbone atoms of the structures with the lowest potential energy (PE) identified within every 2 ps and the structure with the lowest PE searched in the same course of simulation are calculated. For both the HIV IN-LZM and the GCN4-p1 LZM, these rms values decrease with the decrease of PE, which indicates that both structures are closer in conformations as their PEs are moved deeper into the PE well. © 1994 John Wiley & Sons, Inc.     

Differential activities of glucocorticoid-induced leucine zipper protein isoforms

Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.     

Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity

The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.     

Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase

Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.     

repE--the Dictyostelium homolog of the human xeroderma pigmentosum group E gene is developmentally regulated and contains a leucine zipper motif.

We have cloned and characterized the Dictyostelium discoideum repE gene, a homolog of the human xeroderma pigmentosum (XP) group E gene which encodes a UV-damaged DNA binding protein. The repE gene maps to chromosome 4 and it is the first gene identified in Dictyostelium that is homologous to those involved in nucleotide excision repair and their related XP diseases in humans. The predicted protein encodes a leucine zipper motif. The repE gene is not expressed by mitotically dividing cells, and repE mRNA is first detected during the aggregation phase of development when the cells have ceased dividing and replicating genomic DNA. The mRNA level plateaus by the time the developing cells have entered multicellular aggregates and remains at the same steady-state level for the remainder of development. In addition, we have demonstrated that the level of mRNA is very low in developing cells. These observations suggest that repE may play a regulatory role in development. The data indicate that potential developmental roles for XP-related genes can be profitably studied in this system.