GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1͞FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.transcription ͉ factor ͉ gene expression T he erythroid transcription factor GATA-1 has become a paradigm for understanding the control of gene expression by sequence-specific DNA-binding proteins. Numerous studies (reviewed in refs. 1 and 2) have revealed that the function of GATA-1 depends on its ability to bind both DNA and other proteins such as the DNA-binding proteins PU.1, Fli-1 Sp1, and erythroid Krüppel-like factor, as well as coregulators such as LIM-only 2 protein, cyclic AMP response element-binding protein-binding protein, and the zinc-finger protein friend of GATA (FOG). It is thought that different partnerships involving GATA-1 occur at different DNA elements, in different cell types, and at distinct stages of development, and that these regulatory networks contribute to the ability of GATA-1 to direct the complex patterns of gene expression required for the differentiation of blood cells.GATA-family proteins contain two highly conserved trebleclef zinc fingers (ZnFs) domains (3): the N-terminal (NF) and the C-terminal (CF) fingers. These domains mediate both DNA and protein interactions, but despite high sequence similarity to each other (Ͼ50% identity), have quite distinct functions. The CF appears to be the main determinant of DNA-binding and recognizes sequences of the form T͞AGATAA͞G, whereas the NF can participate in binding to tandem T͞AGATAA͞G sites (ref. 4 and references therein) and can independently bind GATC motifs (5). The protein-binding specificities of the two ZnFs also differ; most notably, the NF alone is able to recruit FOG, an Ϸ1,000-aa protein (6) that contains five classic CysCys-His-His (C 2 H 2 ) and four related Cys-Cys-His-Cys (C 2 HC) ZnFs (Fig. 1A).FOG is essential for the normal development of both erythrocytes and megakaryocytes (7) and the interaction of FOG with GATA-1 is indispensable for these events (8). Of the nine ZnFs in FOG (Fig. 1A), four are not found in the tandem arrays that are generally associated with DNA-binding activity. Intriguingly, each of these four isolated domains can independently bind NF, consistent with the emerging view that single fingers can mediate protein-protein interactions (9, 10).Here, we describe the solution structure of...

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a ␤␤␣ fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krü ppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krü ppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.

Homeodomain-only protein (HOP) is an 8-kDa transcriptional corepressor that is essential for the normal development of the mammalian heart. Previous studies have shown that HOP, which consists entirely of a putative homeodomain, acts downstream of Nkx2.5 and associates with the serum response factor (SRF), repressing transcription from SRF-responsive genes. HOP is also able to recruit histone deacetylase (HDAC) activity, consistent with its ability to repress transcription. Unlike other classic homeodomain proteins, HOP does not appear to interact with DNA, although it has been unclear if this is because of an overall divergent structure or because of specific amino acid differences between HOP and other homeodomains. To work toward an understanding of HOP function, we have determined the 3D structure of full-length HOP and used a range of biochemical assays to define the parts of the protein that are functionally important for its repression activity. We show that HOP forms a classical homeodomain fold but that it cannot recognize double stranded DNA, a result that emphasizes the importance of caution in predicting protein function from sequence homology alone. We also demonstrate that two distinct regions on the surface of HOP are required for its ability to repress an SRF-driven reporter gene, and it is likely that these motifs direct interactions between HOP and partner proteins such as SRF- and HDAC-containing complexes. Our results demonstrate that the homeodomain fold has been co-opted during evolution for functions other than sequence-specific DNA binding and suggest that HOP functions as an adaptor protein to mediate transcriptional repression.

Classic zinc finger domains (cZFs) consist of a ␤-hairpin followed by an ␣-helix. They are among the most abundant of all protein domains and are often found in tandem arrays in DNA-binding proteins, with each finger contributing an ␣-helix to effect sequence-specific DNA recognition. Lone cZFs, not found in tandem arrays, have been postulated to function in protein interactions. We have studied the transcriptional co-regulator Friend of GATA (FOG), which contains nine zinc fingers. We have discovered that the third cZF of FOG contacts a coiled-coil domain in the centrosomal protein transforming acidic coiled-coil 3 (TACC3). Although FOG-ZF3 exhibited low solubility, we have used a combination of mutational mapping and protein engineering to generate a derivative that was suitable for in vitro and structural analysis. We report that the ␣-helix of FOG-ZF3 recognizes a C-terminal portion of the TACC3 coiled-coil. Remarkably, the ␣-helical surface utilized by FOG-ZF3 is the same surface responsible for the well established sequence-specific DNA-binding properties of many other cZFs. Our data demonstrate the versatility of cZFs and have implications for the analysis of many as yet uncharacterized cZF proteins.

Because of reports of high levels of interleukin-6 (IL-6) in patients during infection, we studied the role of IL.6 in experimental infection. Mice infected with the facultative intracellular pathogen Listeria monocytogenes displayed high levels of IL-6 in their sera and tissues, particularly the spleen, 1 to 3 days after infection. At this time, the IL-6 titers correlated with bacterial numbers in individual mice and in groups of mice given graded doses of Listeria organisms. However, the presence of IL-6 in serum declined after 4 days, even when a large initial dose of bacteria meant that bacterial numbers were still increasing at this time. Recombinant mouse IL-6 injected intraperitoneally before infection protected mice in a dose-dependent manner. It was effective when given 4 h before infection but not when administration was delayed for 24 h postinfection. It is therefore believed that IL-6 plays a role in early priming of the immune response to infection. Its exact function in this model is being investigated.

Glycosylation is extremely complex, with the potential for a protein to have oligosaccharides attached at multiple sites, and for each site of glycosylation to have multiple structures attached to it. Structural information on the oligosaccharides bound to either asparagine residues (N-linked) or serine and threonine residues (O-linked) requires sensitive, specialised, and complex techniques and equipment. We show here, however, that a large amount of information regarding the glycosylation of glycoproteins can be obtained with common protein techniques such as 1D SDS-PAGE and peptide mass fingerprinting (PMF). Enzymatic deglycosylation in combination with SDS-PAGE and PMF analysis can determine the relative percentage of N-linked carbohydrate on the glycosylated protein, as well as attachment sites of the oligosaccharides.

Injection of recombinant interleukin-6 (IL-6) into mice enhances recovery from infection with Listeria monocytogenes. In this study, the role of IL-6 during primary and secondary Listeria infection was further tested. Neutralization of IL-6 by polyclonal antibody exacerbated primary infection and significantly delayed gamma interferon production by cultured spleen cells. In contrast, administration of anti-IL-6 antibody at the time of secondary infection did not affect the recovery of mice from infection or gamma interferon production, showing that activated T cells are not dependent on IL-6.

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