An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies.

Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this "antigen." Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small ( approximately 180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.

ERN1, a novel ethylene-regulated nuclear protein of Arabidopsis.

Employing differential display of mRNA to investigate the transcriptionally regulated part of the ethylene response pathway in etiolated seedlings of Arabidopsis thaliana, a novel ethylene-regulated nuclear-localized protein, designated ERN1, was identified. ERN1 is one of four genes whose differential expression was confirmed by RNA blot analysis. ERN1 is represented by a single-copy gene in the Arabidopsis genome. Its expression is suppressed by ethylene in wild-type Arabidopsis but not in the ethylene-insensitive etr1-1 mutant. To gain first insight into the biological role of ERN1, a promoter-beta-glucuronidase (GUS) gene fusion was constructed and the expression in various organs from early to late developmental stages was examined. The analysis revealed spatial and temporal expression patterns that correlate with developmental processes known to be affected by ethylene. Evidence is given that the level of expression of ERN1 is regulated through the ethylene signal transduction pathway via CTR1 and EIN3, indicating that ERN1 acts downstream of EIN3.

Analysis of Arabidopsis cDNA that shows homology to the tomato E8 cDNA.

The negative regulatory protein of ethylene synthesis in ripening tomato fruit, E8, is structurally related to the enzyme that catalyzes the last step in ethylene synthesis, 1-aminocyclopropane-1-carboxylate (ACC) oxidase, and to a large family of 2-oxoglutarate-dependent dioxygenases (2-ODD). A cDNA with structural homology to the tomato E8 was isolated from a cDNA library of Arabidopsis thaliana. Sequence analysis showed that this cDNA, 2A6, encodes a protein of 361 amino acids. Southern blot analysis indicated that the corresponding gene is unique in the Arabidopsis genome. The level of the 2A6 transcript was not increased by ethylene in siliques of Arabidopsis, as was E8 in tomato fruits, and was also expressed in etiolated seedlings, leaves, stems and flowers. The 2A6 protein shows three domains that are highly conserved among E8, ACC oxidases, and 2-ODDs.

Functional cis-element sequence requirements for suppression of gene expression by the TNPA protein of the Zea mays transposon En/Spm.

TNPA, one of the two transposition proteins encoded by the En/Spm transposable elements of Zea mays, suppresses the expression of genes that contain an appropriate cis element. Suppression can be monitored in tobacco protoplasts in a transient expression assay as follows. The plant promoter-driven expression of the Escherichia coli-glucuronidase (GUS)-encoding gene, uidA, is repressed in the presence of TNPA if the GUS gene contains a functional cis element in the untranslated RNA leader sequence. Earlier, we found that the minimal cis element is composed of two 12 bp sequences in a tail-to-tail inverted orientation. Each 12 bp sequence is sufficient to bind TNPA in vitro and can be thought of as a half-site in the cis element. Here, we investigated the sequence requirements of the minimal cis element. Our observations support our expectations that a functional cis element must provide a template to which two TNPA molecules can bind in the correct orientation. Sequences within the half-sites can be altered as long as the eight bases that make up the consensus binding sites are not changed. However, we found the following unexpected sequence specificities. Firstly, some changes to the consensus binding sequence can be tolerated in one half-site, as long as the other site matches the consensus. Secondly, although the region between the half-sites can vary in sequence and in length between two and four bases, a thymidine residue is not tolerated directly 5' preceding the second half-site.(ABSTRACT TRUNCATED AT 250 WORDS)

The transposable element En/Spm-encoded TNPA protein contains a DNA binding and a dimerization domain.

The En/Spm-encoded TNPA protein binds to 12-bp DNA sequence motifs that are present in the subtermini of the transposable element. DNA binding of TNPA to monomeric and dimeric forms of the binding motif was analyzed by gel retardation and cross-linking studies. A DNA binding domain at the N-terminal and a dimerization domain at the C-terminal portion of TNPA were localized using deletion derivatives of TNPA. These domains are novel since no apparent homology has been found in the data bases. The stoichiometry of the TNPA-DNA complexes was analyzed. A special complex is formed with a tail-to-tail dimeric DNA binding motif, most probably involving two DNA-bound TNPA molecules that interact via their dimerization domains. In redox reactions the requirement for one or two disulfide bonds for DNA binding of TNPA was shown. The implications of these findings for the excision mechanism of En/Spm are discussed.

Alternative route for biosynthesis of amino sugars in Escherichia coli K-12 mutants by means of a catabolic isomerase.

By inserting a lambda placMu bacteriophage into gene glmS encoding glucosamine 6-phosphate synthetase (GlmS), the key enzyme of amino sugar biosynthesis, a nonreverting mutant of Escherichia coli K-12 that was strictly dependent on exogenous N-acetyl-D-glucosamine or D-glucosamine was generated. Analysis of suppressor mutations rendering the mutant independent of amino sugar supply revealed that the catabolic enzyme D-glucosamine-6-phosphate isomerase (deaminase), encoded by gene nagB of the nag operon, was able to fulfill anabolic functions in amino sugar biosynthesis. The suppressor mutants invariably expressed the isomerase constitutively as a result of mutations in nagR, the locus for the repressor of the nag regulon. Suppression was also possible by transformation of glmS mutants with high-copy-number plasmids expressing the gene nagB. Efficient suppression of the glmS lesion, however, required mutations in a second locus, termed glmX, which has been localized to 26.8 min on the standard E. coli K-12 map. Its possible function in nitrogen or cell wall metabolism is discussed.