Leucine Zipper Dimerization Domain Research Articles

A novel transcription factor negatively regulates antioxidant response, cell wall integrity and virulence in the fungal insect pathogen, Beauveria bassiana.

Genomic data have identified a class of fungal specific transcription factors (FsTFs) that are thought to regulate unique aspects of fungal gene expression, although the functions of many of these proteins remain unknown. Here, a novel FsTF (BbStf1), which features a leucine zipper dimerization domain and a fungal transcription factor regulatory middle homology region, was characterized in Beauveria bassiana, a filamentous insect fungal pathogen. Transcriptional activation and nuclear localization were experimentally confirmed for BbStf1. Disruption of Bbstf1 resulted in increased tolerance to oxidative stress and cell wall perturbation, accompanied by increased peroxidase (POD) and superoxide dismutase (SOD) activities and ratio of reduced/oxidized glutathione (GSH/GSSG), and by thickened cell wall and altered composition. Gene expression profile analysis revealed that transcription patterns of antioxidant enzyme and cell wall integrity-involved genes were altered in the ∆Bbstf1, including some BbStf1-targeted genes clarified with evidence. The ∆Bbstf1 strain displayed greater virulence to Galleria mellonella in the bioassays through both topical infection and intrahaemocoel injection due to more rapid proliferation in the haemocoel as compared to the wild-type strain. Altogether, BbStf1 acts as a negative regulator of antioxidant response, cell wall integrity and virulence in B. bassiana.

Abstract 3850: FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function

Abstract Regulatory T cells (Tregs) are critical for the maintenance of immune tolerance and control of immune responses to foreign agents. Tregs depend upon the transcriptional protein FOXP3 for their ontogeny and genetic orchestration of their suppressive mechanisms. While T cells have the ability to target tumor-associated antigens from many cancer subtypes, these T cells are often suppressed in patients with immunologically “silenced” tumors. The tolerizing function of Tregs can limit effective anti-tumor T cell-mediated killing, even in the presence of immunotherapies, including checkpoint inhibition, and their presence predicts reduced survival in patients with a myriad of cancers. Currently, there exists no means of depleting Tregs in patients without also targeting activated effector T cells. In an effort to overcome this, we have specifically inhibited Tregs by targeting FOXP3 through molecular mimicry using peptides designed to inhibit FOXP3 homodimerization. Homodimerization of FOXP3 is mediated by an antiparallel coiled-coil interaction between the leucine-zipper domains of two FOXP3 proteins. A series of single and double hydrocarbon-stapled alpha-helical (SAH) peptides were synthesized using the leucine zipper dimerization domain (DD) interface of FOXP3 (SAH-FOXP3DDs) as a molecular template. SAH-FOXP3DDs, but not native peptides, specifically bind FOXP3's leucine zipper domain with nanomolar affinity. Unlike stapled peptide point mutant controls, lead SAH-FOXP3DDs also showed dose-dependent inhibition of FOXP3 binding to cognate DNA. Lead SAH-FOXP3DDs are cell permeable and nontoxic to T cells as measured by absence of non-specific LDH-release and apoptosis induction following treatment. Flow cytometric analysis of treated Tregs measured changes in expression of a number of Treg/T cell - associated proteins including increases in FOXP3 and CD127 and decreases in CD25, while no changes were observed in treated conventional CD4+ T cells (Tcons) or with cells treated with point-mutant control peptides. In addition, treated Tregs demonstrated mRNA expression changes of FOXP3-regulated genes including Foxp3, CD127, IL2 and Ctla. Treatment of freshly isolated and in vitro expanded Tregs with lead SAH-FOXP3DD compounds inhibited Treg-mediated suppression of Tcon proliferation in response to CD3/CD28 activation, while no effects were measured with a point-mutant control. We believe that SAH-FOXP3DDs will be powerful molecular probes to further our understanding of FOXP3 transcriptional control. Additionally, they may be a foundation for prototype therapeutics to target Tregs in cancer patients in an effort to amplify the endogenous anti-tumor immune response alone or in combination with other cancer immunotherapies. Citation Format: Rachel Eclov, Marie Fefferman, Ravand Samaeekia, James L. LaBelle. FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3850.

Vibrio parahaemolyticus VtrA is a membrane-bound regulator and is activated via oligomerization.

Vibrio parahaemolyticus is a Gram-negative pathogen that causes food-borne gastroenteritis. A major virulence determinant of the organism is a type III secretion system (T3SS2) encoded on a pathogenicity island, Vp-PAI. Vp-PAI gene expression is regulated by two transcriptional regulators, VtrA and VtrB, whose N-terminal regions share homology with an OmpR-family DNA-binding domain. VtrA activates the gene expression of VtrB, which in turn activates Vp-PAI gene expression; however, the mechanism of this transcriptional activation by VtrA is not well understood. In this study, we determined that VtrA is a membrane protein with a transmembrane (TM) domain, which was required for its transcriptional regulatory activity. Although the N-terminal region of VtrA alone is insufficient for its transcriptional regulatory activity, forced oligomerization using the leucine-zipper dimerization domain of yeast GCN4 conferred transcriptional regulatory activity and a greater affinity for the promoter region of vtrB. A ToxR-based assay demonstrated that VtrA oligomerizes in vivo. We also showed that bile, a host-derived activator of VtrA, induces the oligomerization of VtrA, which requires the C-terminal domain. The promoter region of vtrB contained repetitive T-rich DNA elements, which are important for vtrB transcriptional activation and are conserved among T3SS2-possessing Vibrio species. These findings propose that VtrA is active as oligomers, which may facilitate its N-terminus binding the target DNA, thus enhancing its transcriptional regulatory activity.

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains weres-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4).The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity ofs-tag-BMP-2 in vivo was shown in various experimental animal models.

Altered Expression of Human Smooth Muscle Myosin Phosphatase Targeting (MYPT) Isovariants with Pregnancy and Labor.

BackgroundMyosin light-chain phosphatase is a trimeric protein that hydrolyses phosphorylated myosin II light chains (MYLII) to cause relaxation in smooth muscle cells including those of the uterus. A major component of the phosphatase is the myosin targeting subunit (MYPT), which directs a catalytic subunit to dephosphorylate MYLII. There are 5 main MYPT family members (MYPT1 (PPP1R12A), MYPT2 (PPP1R12B), MYPT3 (PPP1R16A), myosin binding subunit 85 MBS85 (PPP1R12C) and TIMAP (TGF-beta-inhibited membrane-associated protein (PPP1R16B)). Nitric oxide (NO)-mediated smooth muscle relaxation has in part been attributed to activation of the phosphatase by PKG binding to a leucine zipper (LZ) dimerization domain located at the carboxyl-terminus of PPP1R12A. In animal studies, alternative splicing of PPP1R12A can lead to the inclusion of a 31-nucleotide exonic segment that generates a LZ negative (LZ-) isovariant rendering the phosphatase less sensitive to NO vasodilators and alterations in PPP1R12ALZ- and LZ+ expression have been linked to phenotypic changes in smooth muscle function. Moreover, PPP1R12B and PPP1R12C, but not PPP1R16A or PPP1R16B, have the potential for LZ+/LZ- alternative splicing. Yet, by comparison to animal studies, the information on human MYPT genomic sequences/mRNA expressions is scant. As uterine smooth muscle undergoes substantial remodeling during pregnancy we were interested in establishing the patterns of expression of human MYPT isovariants during this process and also following labor onset as this could have important implications for determining successful pregnancy outcome.ObjectivesWe used cross-species genome alignment, to infer putative human sequences not available in the public domain, and isovariant-specific quantitative PCR, to analyse the expression of mRNA encoding putative LZ+ and LZ- forms of PPP1R12A, PPP1R12B and PPP1R12C as well as canonical PPP1R16A and PPP1R16B genes in human uterine smooth muscle from non-pregnant, pregnant and in-labor donors.ResultsWe found a reduction in the expression of PPP1R12A, PPP1R12BLZ+, PPP1R16A and PPP1R16B mRNA in late pregnancy (not-in-labor) relative to non-pregnancy. PPP1R12ALZ+ and PPP1R12ALZ- mRNA levels were similar in the non-pregnant and pregnant not in labor groups. There was a further reduction in the uterine expression of PPP1R12ALZ+, PPP1R12CLZ+ and PPP1R12ALZ- mRNA with labor relative to the pregnant not-in-labor group. PPP1R12A, PPP1R12BLZ+, PPP1R16A and PPP1R16B mRNA levels were invariant between the not in labor and in-labor groups.ConclusionsMYPT proteins are crucial determinants of smooth muscle function. Therefore, these alterations in human uterine smooth muscle MYPT isovariant expression during pregnancy and labor may be part of the important molecular physiological transition between uterine quiescence and activation.

Gene Expression and Transcription Factor Profiling Reveal Inhibition of Transcription Factor cAMP-response Element-binding Protein by γ-Herpesvirus Replication and Transcription Activator

Herpesvirus replication involves the expression of over 80 viral genes in a well ordered sequence, leading to the production of new virions. Viral genes expressed during the earliest phases of replication often regulate both viral and cellular genes. Therefore, they have the potential to bring about dramatic functional changes within the cell. Replication and transcription activator (RTA) is a potent immediate early transcription activator of the gamma-herpesvirus family. This family includes Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus, human pathogens associated with malignancy. Here we combine gene array technology with transcription factor profiling to identify the earliest DNA promoter and cellular transcription factor targets of RTA in the cellular genome. We find that expression of RTA leads to both activation and inhibition of distinct groups of cellular genes. The identity of the target genes suggests that RTA rapidly changes the cellular environment to counteract cell death pathways, support growth factor signaling, and also promote immune evasion of the infected cell. Transcription factor profiling of the target gene promoters highlighted distinct pathways involved in gene activation at specific time points. Most notable throughout was the high level of cAMP-response element-binding protein (CREB)-response elements in RTA target genes. We find that RTA can function as either an activator or an inhibitor of CREB-response genes, depending on the promoter context. The association with CREB also highlights a novel connection and coordination between viral and cellular "immediate early" responses.

Unfolding of bZIP dimers formed by the ATF-2 and c-Jun transcription factors is not a simple two-state transition

The varied selectivity of bZIP transcription factors stems from the fact that they are dimers consisting of two not necessarily identical subunits held together by a leucine zipper dimerization domain. Determining their stability is therefore important for understanding the mechanism of formation of these transcription factors. The most widely used approach for this problem consists of observing temperature-induced dissociation of the bZIPs by any means sensitive to their structural changes, particularly optical methods. In calculating thermodynamic characteristics of this process from such data it is usually assumed that it represents a two-state transition. However, scanning micro-calorimetric study of the temperature-induced unfolding/dissociation of the three bZIPs formed by the ATF-2 and c-Jun proteins, i.e. the two homodimers (ATF-2/ATF-2) and (c-Jun/c-Jun) and the heterodimer (ATF-2/c-Jun), showed that this process does not represent a two-state transition, as found previously with the GCN4 homodimeric bZIP protein. This raises doubt about all indirect estimates of bZIP thermodynamic characteristics based on analysis of their optically-observed temperature-induced changes.

Design, selection, and characterization of a split chorismate mutase

Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.

PNA zipper as a dimerization tool: Development of a bZip mimic

The article describes the use of a PNA duplex (PNA zipper) as a tool to dimerize or bring in close proximity two polypeptides or protein domains. The amino acid sequence to be dimerized is covalently bound to complementary PNA sequences. Annealing of the PNA strands results in dimer formation. To test the ability of the "PNA-zipper" as a dimerization tool, we designed a GCN4 mimetic, where the leucine-zipper dimerization domain was replaced by the PNA zipper, whereas the basic DNA-binding domain was covalently attached to the PNA. The molecule was assembled by chemical ligation of the peptide corresponding to the DNA-binding domain of GCN4 modified with a succinyl thioester with two complementary PNAs harboring a cysteine residue. Electromobility-shift experiments show the ability of the PNA zipper-GCN4 to bind selected DNA duplexes. The PNA zipper-GCN4 binds both the TRE and CRE DNA sites, but it does not bind TRE and CRE mutants containing even a single base mutation, as the native GCN4. The ability to fold upon complexation with DNA was investigated by CD. A good correlation between the ability of the PNA zipper-GCN4 to fold into alpha helices and the ability to bind DNA was found.

Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K d 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

Self‐assembly of coiled‐coil tetramers in the 1.40 Å structure of a leucine‐zipper mutant

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.

Erythroid-Specific Apoptosis Induced by Jun Transcription Factors Employs a Novel Pathway Involving Recruitment of COP1 and Itch Ubiquitin Ligases.

Upregulation of the transcription factor c-Jun has been identified in acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, and in iron deficiency. These conditions all share as a common feature an impairment of erythropoiesis, in some instances manifested by apoptosis of erythroid progenitors. Depending on cellular context, c-Jun may promote either survival or apoptosis, the latter occurring primarily through regulation of target genes such as Bim and FasL. Transcriptional activation by its kinase JNK typically enhances the apoptotic effects of c-Jun. In primary human erythroid progenitors, we have identified a novel Jun-mediated apoptotic pathway that does not depend on transcriptional activation and is associated with retrograde inhibition of JNK signaling. To determine the nature of this pathway, we have employed a system of retroviral transduction of human CD34+ peripheral blood mobilized progenitors, which undergo culture in serum free erythroid medium containing erythroipoietin and stem cell factor. Wild type c-Jun and JunB, as well as c-Jun mutants lacking transactivation and DNA binding functions, all efficiently induced caspase-dependent apoptosis in early erythroid precursors within the CD36-positive GPA-dim compartment. By contrast, transduced cells undergoing megakaryocytic and myeloid differtentiation, as indicated by CD41 and CD13 expression, showed minimal apoptosis regardless of construct. Deletion or replacement of the c-Jun leucine zipper (LZ) dimerization domain completely eliminated the induction of erythroid apoptosis. Introduction of an LZ point mutation E281K, which alters dimerization specificity, also eliminated induction of apoptosis. These results demonstrated the dispensibility of direct target gene regulation but indicated an absolute requirement for specific LZ protein-protein interactions. Because JunD showed relatively inefficient apoptotic induction, we considered a role for recruitment of ubiquitin ligases such as Itch, which selectively binds c-Jun and JunB. Indeed, the c-Jun Y170F mutation, which ablates the PPXY binding site for Itch (and possibly other WW-domain ubiquitin ligases), completely abrogated apoptosis. Surprisingly, the c-Jun mutant VP232/233AA, which eliminates recruitment of the COP1 RING finger ubiquitin ligase, also manifested major defects in apoptosis induction. Biochemical analysis of the transduced cells showed that induction of apoptosis positively correlated with repression of JNK phosphorylation. Wild type c-Jun, JunB, and mutants that retained induction of apoptosis blocked JNK phosphorylation. All mutants tested that were defective in apoptosis induction caused an enhancement of JNK phosphorylation. We therefore postulate that this novel pathway of erythroid apoptosis results from c-Jun nucleation of a protein complex involving an LZ partner and multiple ubquitin ligases. Assembly of this complex then leads to blockade of JNK phosphorylation and induction of apoptosis.

Self-assembly of receptor/signaling complexes in bacterial chemotaxis.

Escherichia coli chemotaxis is mediated by membrane receptor/histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, lzTar(C), that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from lzTar(C). Thus, the organization of these receptor/signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo.

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

Experimental identification of homodimerizing B-ZIP families in Homo sapiens

B-ZIP transcription factors dimerization is mediated by a parallel coiled-coil termed the leucine zipper. We have evaluated the dimerization specificity of the seven coiled-coil B-ZIP proteins (ATF6, XBP, LZIP, NFIL3, TEF, CREB, and C/EBPα) with themselves and each other. To do this, we designed dominant negative proteins, termed A-ZIPs, that contain the leucine zipper dimerization domain of a B-ZIP protein and an acidic amphipathic N-terminal extension. The A-ZIPs heterodimerize with B-ZIP proteins in a leucine zipper-dependent manner. The acidic N-terminal extension is hypothesized to form an heterodimeric coiled-coil structure with the basic region, essentially zippering the leucine zipper into the basic region. We now present a new acidic extension design that stabilizes heterodimerization with B-ZIP proteins up to 11 kcal mol −1. We have used these A-ZIP proteins in a competition EMSA to evaluate which A-ZIP can prevent DNA binding of which B-ZIP domain. Inhibition of DNA binding is interpreted to indicate that the A-ZIP is forming a heterodimer with the B-ZIP domain and thus prevents the B-ZIP from binding to DNA. All leucine zippers examined can homodimerize and two pairs (CREB & NFIL3 and ATF6 & XBP) can heterodimerize. We discuss these results with reference to the amino acid sequence of the leucine zipper region. These A-ZIP reagents may be of value in biological systems to inhibit the DNA binding and transcriptional potential of specific B-ZIP families.

Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hydrophobic Heptad Repeat

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

Identification of the Interface between cGMP-dependent Protein Kinase Iβ and Its Interaction Partners TFII-I and IRAG Reveals a Common Interaction Motif

Protein-protein interactions have emerged as an important mechanism providing for specificity in cellular signal transduction. Two splice variants of type I cGMP-dependent protein kinase (PKG Ialpha and Ibeta) differ only in their N-terminal approximately 100 amino acids, which mediate binding to different target proteins. PKG Ibeta, but not Ialpha, binds to the general transcriptional regulator TFII-I and the inositol 1,4,5-trisphosphate receptor-associated PKG substrate IRAG. Using a combination of site-directed mutagenesis and in vitro binding assays, we identified a group of acidic amino acids in the N-terminal leucine zipper dimerization domain of PKG Ibeta required for its binding to both TFII-I and IRAG. Small clusters of basic amino acids in possible alpha-helical regions in TFII-I and IRAG were found to mediate their interaction with PKG Ibeta. Mutation of two negatively charged residues in the PKG Ibeta leucine zipper (D26K/E31R) to positively charged residues, found in corresponding positions in PKG Ialpha, completely abrogated binding to TFII-I and IRAG without disrupting PKG dimerization. Mutation of specific basic residues in TFII-I or IRAG abolished binding of the full-length proteins to PKG Ibeta in intact cells. Based on these results, we propose a model for specific PKG Ibeta interaction with target proteins.

Dual DNA recognition codes of a short peptide derived from the basic leucine zipper protein EmBP1

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a β-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5′-GCCACGTGGC-3′ and the native GCN4 binding 5′-ATGACGTCAT-3′ sequences with almost equal affinity in the α-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5′-ATGAC-3′ in the α-helical conformation, but it neither shows affinity nor helix formation with 5′-GCCAC-3′. Because native EmBP1 forms 100 times more stable complex with 5′-GCCACGTGGC-3′ over 5′-ATGACGTCAT-3′, our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.

Complexity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus.

Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), we generated mice deficient in this transcription factor. We performed targeted disruption of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(-/-) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. We observed no differences in various blood parameters and chemistry values. We infected wild-type and Nrf3(-/-) mice with acute lymphocytic choriomeningitis virus and found no differences in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, we generated mice deficient in multiple CNC factors. Contrary to our expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(-/-)/Nrf2(-/-) and Nrf3(-/-)/p45(-/-) mice. We hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice.

Structure of a Designed Dimeric Zinc Finger Protein Bound to DNA,

Proteins that employ dimerization domains to bind cooperatively to DNA have a number of potential advantages over monomers with regards to gene regulation. Using a combination of structure-based design and phage display, a dimeric Cys(2)His(2) zinc finger protein has been created that binds cooperatively to DNA via an attached leucine zipper dimerization domain. This chimera, derived from components of Zif268 and GCN4, displayed excellent DNA-binding specificity, and we now report the 1.5 A resolution cocrystal structure of the Zif268-GCN4 homodimer bound to DNA. This structure shows how phage display has annealed the DNA binding and dimerization domains into a single functional unit. Moreover, this chimera provides a potential platform for the creation heterodimeric zinc finger proteins that can regulate a desired target gene through cooperative DNA recognition.