Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin.

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

A dual role for glucocorticoid-induced leucine zipper in glucocorticoid function: tumor growth promotion or suppression?

Glucocorticoids (GCs), important therapeutic tools to treat inflammatory and immunosuppressive diseases, can also be used as part of cancer therapy. In oncology, GCs are used as anticancer drugs for lymphohematopoietic malignancies, while in solid neoplasms primarily to control the side effects of chemo/radiotherapy treatments. The molecular mechanisms underlying the effects of GCs are numerous and often overlapping, but not all have been elucidated. In normal, cancerous, and inflammatory tissues, the response to GCs differs based on the tissue type. The effects of GCs are dependent on several factors: the tumor type, the GC therapy being used, the expression level of the glucocorticoid receptor (GR), and the presence of any other stimuli such as signals from immune cells and the tumor microenvironment. Therefore, GCs may either promote or suppress tumor growth via different molecular mechanisms. Stress exposure results in dysregulation of the hypothalamic-pituitary-adrenal axis with increased levels of endogenous GCs that promote tumorigenesis, confirming the importance of GCs in tumor growth. Most of the effects of GCs are genomic and mediated by the modulation of GR gene transcription. Moreover, among the GR-induced genes, glucocorticoid-induced leucine zipper (GILZ), which was cloned and characterized primarily in our laboratory, mediates many GC anti-inflammatory effects. In this review, we analyzed the possible role for GILZ in the effects GCs have on tumors cells. We also suggest that GILZ, by affecting the immune system, tumor microenvironment, and directly cancer cell biology, has a tumor-promoting function. However, it may also induce apoptosis or decrease the proliferation of cancer cells, thus inhibiting tumor growth. The potential therapeutic implications of GILZ activity on tumor cells are discussed here.

Glucocorticoid-induced leucine zipper (GILZ) in immuno suppression: master regulator or bystander?

Induction of glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids has been reported to be essential for their anti-inflammatory actions. At the same time, GILZ is actively downregulated under inflammatory conditions, resulting in an enhanced pro-inflammatory response. Two papers published in the recent past showed elevated GILZ expression in the late stage of an inflammation. Still, the manuscripts suggest seemingly contradictory roles of endogenous GILZ: one of them suggested compensatory actions by elevated corticosterone levels in GILZ knockout mice, while our own manuscript showed a distinct phenotype upon GILZ knockout in vivo. Herein, we discuss the role of GILZ in inflammation with a special focus on the influence of endogenous GILZ on macrophage responses and suggest a cell-type specific action of GILZ as an explanation for the conflicting results as presented in recent reports.

A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.

Silencing of the glucocorticoid-induced leucine zipper improves the immunogenicity of clinical-grade dendritic cells.

BACKGROUND: The maturation cocktail composed of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α and prostaglandin E2 is considered the "gold standard" for inducing the maturation of dendritic cells (DCs) for use in cancer immunotherapy. Nevertheless, although this maturation cocktail induces increased expression of several activation markers, such as CD83, the co-stimulation molecules CD80, CD86 and CD40 and the chemokine receptor involved in DC homing in lymph nodes CCR7, the DC immune stimulatory function in vivo contrasts with this mature phenotype, and good clinical outcomes in patients with cancer treated with DC-based vaccines remain rare. METHODS: Phenotypic characterization of the immunosuppressive status of DCs differentiated from peripheral blood mononuclear cells of healthy volunteers and matured with the "gold standard" cocktail was performed. Glucocorticoid-induced leucine zipper (GILZ) targeting small interfering RNA (siRNA) was electroporated into DCs after maturation to increase their immunogenicity. RESULTS: The mature phenotype of DCs treated for 48 h with this cocktail was associated with the expression of several immunosuppressive regulators, including programmed cell death 1 ligand 1 (PD-L1), IL-10 and GILZ. Electroporation is a very efficient and safe way to deliver siRNA into DCs (80% of DCs receive at least one molecule of siRNA). Silencing GILZ in clinical-grade DCs by siRNA leads to a decrease of the PD-L1 expression associated with an increase in their IL-12 secretion and T-cell induction capability. CONCLUSIONS: GILZ silencing is a promising approach to achieving complete clinical-grade DC maturation and avoiding the immunosuppressive effects of the maturation cocktail on DCs intended for clinical use.

Domain requirements for the diverse immune regulatory functions of foxp3.

Foxp3 is responsible for the major immunological features of Treg cells, including hypoproliferation in vitro, immune suppression of conventional T cells and resistance to Th2 cell differentiation. In addition to the Forkhead domain, the Foxp3 protein contains the N-terminal, zinc finger and leucine zipper domains. To understand how these domains contribute to Foxp3 functions, we systematically compared the roles of these domains in determining the 3 major immunological features of Treg cells. We designed a bridge-mediated mutagenesis method to generate Foxp3 mutants with complete deletion of each of the domains. CD4 T cells expressing the Foxp3 mutant with deletion of the N-terminal, leucine zipper or the forkhead domain showed robust TCR dependent proliferation in vitro, differentiated into Th2 cells, and lost immune suppressive activities in vitro and in vivo, demonstrating a complete loss of all 3 functions of Foxp3. In contrast, deletion of the zinc finger domain only partially impaired these functions of Foxp3. This result suggests that mutations in the zinc finger domain could lead to nonlethal autoimmune and allergic diseases, in which reduction rather than complete loss of Foxp3 functions is expected. In any case, deletion of a particular domain showed similar effects on all 3 functions of Foxp3. Therefore defining each of the immunological features of Treg cells requires intact Foxp3 proteins.

Induction of the glucocorticoid-induced leucine zipper gene limits the efficacy of dendritic cell vaccines.

Dendritic cell (DC) vaccines have shown great promise in generating antitumor immune responses but have generally fallen short of producing durable cures. Determining mechanisms by which these vaccines fail will provide one strategy toward improving their success. Several manipulations of DCs have improved their migration and longevity, but the immune inhibitory environment surrounding tumors provides a powerful suppressive influence. To determine the mechanisms by which DCs at the site of the tumor convert to a suppressive phenotype, we evaluated pathways in DCs that become expressed at the tumor site. Our results revealed that tumors lead to induction of the glucocorticoid-induced leucine zipper (GILZ) gene in DCs, and that this gene is critical for the development of tumor-induced tolerance of both DCs and T cells. Previous data suggested that GILZ is a pivotal gene in the balance between activation and tolerance of DCs. Our new data show that GILZ is highly upregulated in DCs in the tumor microenvironment in vivo and that blockade of this gene in DC vaccines significantly improves long-term survival. These results suggest that GILZ may be an ideal candidate gene to target for novel immune-based tumor therapies.

CD80 blockade enhance glucocorticoid-induced leucine zipper expression and suppress experimental autoimmune encephalomyelitis.

Designing mimetic of the interface functional groups of known receptor-ligand complexes is an attractive strategy for developing potential therapeutic agents that interfere with target protein-protein interactions. The CD80/CD86-CD28/CD152 costimulatory interactions transmit signals for CD4(+) T cell activation and suppression and are critically involved in the initiation, progression, and reactivation of the immunopathology in multiple sclerosis. Differences in the pattern, levels, and kinetics of expression of CD80/CD86 molecules in conjunction with differences in the strength of the signals delivered upon binding CD28 or CD152 determine the outcome of the immune response. A temporal up-regulation of surface expression of CD80 relative to CD86 on APCs and CNS-infiltrating cells has been shown to correlate with disease progression in experimental autoimmune encephalomyelitis an animal model for multiple sclerosis. Hence blockade of the CD80 costimulatory axis has therapeutic potential in multiple sclerosis. In this study, we report the efficacy of a novel CD80-blocking agent CD80-competitive antagonist peptide (CD80-CAP) in suppressing clinical disease and relapse in experimental autoimmune encephalomyelitis. The CD80-CAP mediates protection by inhibiting proinflammatory cytokines and skewing toward anti-inflammatory response presumably by enhancing the expression of glucocorticoid-induced leucine zipper in activated CD4(+) T cells.

Antibodies purified from sera of HIV-1-infected patients by affinity on the heptad repeat region 1/heptad repeat region 2 complex of gp41 neutralize HIV-1 primary isolates.

OBJECTIVE: The objective of this paper was to evaluate the presence and the neutralizing activity of antibodies directed against the complex formed between the two heptad repeat regions (HR1 and HR2) of HIV-1 gp41 in sera of HIV-1-infected patients. RESEARCH DESIGNS AND METHODS: The HR1 region was represented by the peptide N36 and the maltose-binding protein (MBP)-HR1, the HR2 region by the peptide C34 and MBP44. Antibodies directed to the HR1/HR2 complex were purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed onto a resin. RESULTS: First, we demonstrated that human monoclonal antibodies, which are directed specifically to the HR1/HR2 complex recognized in enzyme-linked immunosorbent assay the MBP-HR1/C34 and MBP44/N36 mixtures but not the proteins or the peptides individually. We investigated the ability of 50 sera of HIV-1-infected patients to react with the MBP-HR1/C34 and MBP44/N36 complexes. We found that the majority of sera of HIV-1-infected patients recognized the HR1/HR2 complexes but not or to a lower extent the proteins or the peptides individually. Antibodies purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed to a resin neutralized different primary HIV-1 isolates. CONCLUSION: The presence of antibodies directed to the HR1/HR2 complex in sera of HIV-infected patients highlights the immunogenic character of the complex, whereas the neutralizing activity of these antibodies suggests that immunogens representing HIV-1 HR1/HR2 complexes might be used in anti-HIV vaccine.

Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein.

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200-239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody-antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200-239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient-derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI-TOF-MS demonstrate that the antibody recognition is dependent on a partly alpha-helical fold within the putative leucine zipper of the 200-239 aa stretch and that the two human anti-p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1-like antibodies.

Cloning and characterization of two human Ro52-specific monoclonal autoantibodies directed towards a domain associated with congenital heart block.

Autoantibodies against amino acid 200-239 (p200) in the predicted leucine zipper region of the Ro52 protein are associated with congenital heart block, a potentially fatal condition that may affect fetuses of women with Ro52 autoantibodies. To allow detailed studies of the antibodies associated with congenital heart block, B-cell derived combinatorial antibody libraries from patients were screened for Ro52 and p200 specific antibody clones. Two human monoclonal anti-p200 antibody fragments, S3A8 and M4H1, were isolated and analysed with regard to VHand VL gene utilization, somatic mutations and binding properties. Both identified clones recognized recombinant and native intact Ro52, and reacted only with p200 in a set of related Ro52 peptides. The specificity and affinity was confirmed by biosensor measurements. Structural analysis of overlapping peptides revealed increased helicity in the p200 peptide compared to non-recognized peptides, indicating epitope conformation as essential for antibody binding. Both monoclonals produced punctate nuclear and diffuse cytoplasmic staining in human and mouse cell lines. The identified antibodies, which react specifically with the leucine zipper structure of Ro52, will be valuable in further exploration of the mechanisms operating during development of Ro52 antibody-associated congenital heart block.

Four reasons to consider a novel class of innate immune molecules in the oral epithelium.

An expanding number of innate immune molecules occupy the "epithelial frontier". This review introduces a recently recognized class of mammalian proteins with similarity to PLUNC (palate, lung and nasal epithelium clone), which is itself related to the host defense protein BPI (bactericidal/permeability-increasing protein). Four emerging lines of evidence unite the PLUNC-like proteins: conserved genetic structure, epithelial expression, three-dimensional protein similarity, and a physiological response to injury or inflammation. By analogy to known proteins of the innate immune system, an emerging hypothesis for this family is that they act as sensors of Gram-negative bacteria in the oral cavity, among other areas.

Temperature effect on IgE binding to CD23 versus Fc epsilon RI.

A chimeric soluble CD23, consisting of the extracellular domain of mouse CD23 and a modified leucine zipper (lz-CD23), has been shown to inhibit IgE binding to the FcepsilonRI. A similar human CD23 construct was also shown to inhibit binding of human IgE to human FcepsilonRI. In both systems, the inhibition was found to be temperature dependent; a 10-fold molar excess of lz-CD23 gave 90-98% inhibition at 4 degrees C, dropping to 20-30% inhibition at 37 degrees C. Surface plasmon resonance analysis of lz-CD23 binding to an IgE-coated sensor chip suggested that the effective concentration of lz-CD23 was lower at the higher temperatures. Analysis of (125)I-IgE binding to CD23(+)-Chinese hamster ovary cells also indicated that increased temperature resulted in a lower percentage of IgE capable of interacting with CD23. In contrast, IgE interacts more effectively with FcepsilonRI(+)-rat basophilic leukemia cells at 37 degrees C compared with 4 degrees C. The results support the concept that the open and closed IgE structures found by crystallography interact differently with the two IgE receptors and suggest that temperature influences the relative percentage of IgE in the respective structural forms. Changes in CD23 oligomerization also plays a role in the decreased binding seen at physiological temperatures.

Acceleration of human neutrophil apoptosis by TRAIL.

Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive program of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well-documented and TNF-alpha also has a transient proapoptotic effect. We have studied the role of the death receptor ligand TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2, and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine zipper-tagged form of TRAIL, which mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors, specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. No additional proapoptotic effect of leucine zipper TRAIL was identified following TRAIL treatment of neutrophils in the presence of gliotoxin, an inhibitor of NF-kappaB, suggesting TRAIL does not activate NF-kappaB in human neutrophils. TRAIL treatment of human neutrophils did not induce a chemotactic response. The susceptibility of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation.

Synthesis of glucocorticoid-induced leucine zipper (GILZ) by macrophages: an anti-inflammatory and immunosuppressive mechanism shared by glucocorticoids and IL-10.

Glucocorticoids and interleukin 10 (IL-10) prevent macrophage activation. In murine lymphocytes, glucocorticoids induce expression of glucocorticoid-induced leucine zipper (GILZ), which prevents the nuclear factor kappaB (NF-kappaB)-mediated activation of transcription. We investigated whether GILZ could account for the deactivation of macrophages by glucocorticoids and IL-10. We found that GILZ was constitutively produced by macrophages in nonlymphoid tissues of humans and mice. Glucocorticoids and IL-10 stimulated the production of GILZ by macrophages both in vitro and in vivo. Transfection of the macrophagelike cell line THP-1 with the GILZ gene inhibited the expression of CD80 and CD86 and the production of the proinflammatory chemokines regulated on activation normal T-cell expressed and secreted (CCL5) and macrophage inflammatory protein 1alpha (CCL3). It also prevented toll-like receptor 2 production induced by lipopolysaccharide, interferongamma, or an anti-CD40 mAb, as well as NF-kappaB function. In THP-1 cells treated with glucocorticoids or IL-10, GILZ was associated with the p65 subunit of NF-kappaB. Activated macrophages in the granulomas of patients with Crohn disease or tuberculosis do not produce GILZ. In contrast, GILZ production persists in tumor-infiltrating macrophages in Burkitt lymphomas. Therefore, GILZ appears to play a key role in the anti-inflammatory and immunosuppressive effects of glucocorticoids and IL-10. Glucocorticoid treatment stimulates GILZ production, reproducing an effect of IL-10, a natural anti-inflammatory agent. The development of delayed-type hypersensitivity reactions is associated with the down-regulation of GILZ gene expression within lesions. In contrast, the persistence of GILZ gene expression in macrophages infiltrating Burkitt lymphomas may contribute to the failure of the immune system to reject the tumor.

Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappaB.

Previously a novel gene was identified that encodes a glucocorticoid-induced leucine zipper (GILZ) whose expression is up-regulated by dexamethasone. This study analyzed the role of GILZ in the control of T-cell activation and its possible interaction with nuclear factor kappaB (NF-kappaB). Results indicate that GILZ inhibits both T-cell receptor (TCR)-induced interleukin-2/interleukin-2 receptor expression and NF-kappaB activity. In particular, GILZ inhibits NF-kappaB nuclear translocation and DNA binding due to a direct protein-to-protein interaction of GILZ with the NF-kappaB subunits. Moreover, GILZ-mediated modulation of TCR-induced responses is part of a circuit because TCR triggering down-regulates GILZ expression. These results identify a new molecular mechanism involved in the dexamethasone-induced regulation of NF-kappaB activity and T-cell activation. (Blood. 2001;98:743-753)

CHOP, a basic leucine zipper transcriptional factor, contributes to the antiproliferative effect of IL-1 on A375 human melanoma cells through augmenting transcription of IL-6.

Interleukin-1 (IL-1) inhibits the proliferation of A375 human melanoma cells. We have demonstrated previously that p38 mitrogen-activated protein kinase (MAPK) mediated the antiproliferative effect of IL-1 partially through the downregulation of activity and protein level of ornithine decarboxylase (ODC). In this study, we investigated the role of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the p38 MAPK target transcriptional factors. The mRNA level of CHOP was not affected by IL-1 treatment in A375-6 cells. Unexpectedly, CHOP was constitutively phosphorylated, and IL-1 or p38 MAPK inhibitor, SB203580, did not affect the phosphorylation level. However, A375-6 cells exhibited enhanced sensitivity to IL-1 by transfecting CHOP expression plasmid and reduced sensitivity to IL-1 by antisense CHOP mRNA expression plasmid. Furthermore, CHOP appeared to regulate positively IL-6 production at the transcriptional level. The experiments using CHOP muteins revealed that dimerization ability - but not p38 MAPK-dependent phosphorylation or DNA binding activity - is important for the IL-6 inducing activity of CHOP. These results indicate that CHOP contributes to the IL-1 growth-inhibitory signal through augmenting IL-6 production.

Autoantibody to the leucine zipper region of 52 kDa Ro/SSA binds native 60 kDa Ro/SSA: identification of a tertiary epitope with components from 60 kDa Ro/SSA and 52 kDa Ro/SSA.

Anti-Ro (or SSA) is found in the sera of patients with autoimmune rheumatic illnesses. All patients with anti-Ro defined by precipitation bind a 60 000 Da antigen (60 kDa Ro), whereas some patients also bind a 52 000 Da molecule (52 kDa Ro). In general, antibody binding is directed against native 60 kDa Ro and denatured 52 kDa Ro. The mechanism by which anti-52 kDa Ro arises in the setting of anti-60 kDa Ro is unknown. Conflicting data exist as to the existence of a physical interaction between the two proteins in cells and as to cross-reacting antibodies. Antibodies were affinity purified from a peptide within the leucine zipper region of 52 kDa Ro. These purified antibodies binding the 197-207 peptide from 52 kDa Ro (anti-52LZ) bound native 60 kDa Ro as well as denatured 52 kDa Ro. In addition, anti-52LZ also bound up to four regions from the sequence of 60 kDa Ro and a single conformational epitope of 60 kDa Ro. Thus, these primary sites represent components of the tertiary epitope. We hypothesized that if this was the case, these peptides making up a tertiary epitope would show molecular interaction. In fact, peptides from 60 kDa Ro have a molecular interaction with the 52 kDa Ro peptide as well as full-length 52 kDa Ro when assessed by surface plasmon resonance. The leucine-zipper region peptide from 52 kDa Ro bound three of the four peptides from 60 kDa Ro. These data suggest that these two molecular species, 60 and 52 kDa Ro, form a conformational epitope. This relationship may explain why anti-52 kDa Ro is found in association with anti-60 kDa Ro.

Repression of IL-2 promoter activity by the novel basic leucine zipper p21SNFT protein.

IL-2 is the major autocrine and paracrine growth factor produced by T cells upon T cell stimulation. The inducible expression of IL-2 is highly regulated by multiple transcription factors, particularly AP-1, which coordinately activate the promoter. Described here is the ability of the novel basic leucine zipper protein p21SNFT to repress AP-1 activity and IL-2 transcription. A detailed analysis of the repression by p21SNFT repression on the IL-2 promoter distal NF-AT/AP-1 site demonstrates that it can bind DNA with NF-AT and Jun, strongly suggesting that it represses NF-AT/AP-1 activity by competing with Fos proteins for Jun dimerization. The importance of this repression is that p21SNFT inhibits the trans-activation potential of protein complexes that contain Jun, thereby demonstrating an additional level of control for the highly regulated, ubiquitous AP-1 transcription factor and the IL-2 gene.

Antigen recognition by conformational selection.

Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an 'induced fit', which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an 'induced fit' requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross-reacts with the wild-type dimeric leucine zipper. Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.