Mouse deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.     

Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer-promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair-rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions.     

Pathogenesis of experimentally induced rabies in domestic ferrets

Isl-1 is a member of a family of Homeodomains containing proteins that possess an N-terminal pair of zinc binding LIM domains. The Isl-1 gene in rat codes for a protein that binds to the insulin gene enhancer and is also involved in regulation of amylin and proglucagon genes. A DNA sequence coding for 66 amino acid residues containing the C-terminal homeodomain fragment of Isl-1 was expressed as a soluble protein in Escherichia coli. Here, we describe a procedure which allows the rapid native purification of recombinant homeodomain protein fused to an N-terminal tag of six histidines. The purified homeodomain showed DNA-binding activity to its cognate DNA sequence. An enhanced binding activity is observed in the presence of a reducing agent in electrophoretic mobility shift assays. The DNA binding was further characterized by circular dichroism spectroscopy. Addition of DNA to the homeodomain did not change the overall secondary structure content, but the thermal and chemical denaturing profiles were altered. A stabilization of the secondary structure was observed upon DNA binding. The free energy of unfolding at 23 degrees C was 7 kJ mol(-1) in absence of DNA and 29 kJ mol(-1) in the presence of DNA.     

Specificity of LIM domain interactions with receptor tyrosine kinases

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.     

Structure and intramodular dynamics of the amino-terminal LIM domain from quail cysteine- and glycine-rich protein CRP2

Members of the cysteine and glycine-rich protein (CRP) family (CRP1, CRP2, and CRP3) contain two zinc-binding LIM domains, LIM1 and LIM2, and are implicated in diverse cellular processes linked to differentiation, growth control and pathogenesis. The solution structure of an 81-amino acid recombinant peptide encompassing the amino-terminal LIM1 domain of quail CRP2 has been determined by 2D and 3D homo- and heteronuclear NMR spectroscopy. The LIM1 domain consists of two zinc binding sites of the CCHC and the CCCC type, respectively, which both contain two orthogonally arranged antiparallel beta-sheets and which are packed together by a hydrophobic core composed of residues from the zinc finger loop regions. The CCCC zinc finger is followed by a short alpha-helical stretch. The structural analysis revealed that the global fold of LIM1 closely resembles the recently determined solution structures of the carboxyl-terminal LIM2 domains of quail CRP2 and chicken CRP1, and that LIM1 and LIM2 are independently folded structural and presumably functional domains of CRP proteins. To explore the dynamical properties of CRP proteins, we have used 15N relaxation values (T1, T2, and nuclear Overhauser effect (NOE) to describe the dynamical behavior of a LIM domain. A model-free analysis revealed local variations in mobility along the backbone of the quail CRP2 LIM1 motif. Slow motions are evident in turn regions located between the various antiparallel beta-sheets or between their strands. By use of an extended motional model, fast backbone motions were detected for backbone amide NH groups of hydrophobic residues located in the core region of the LIM1 domain. These findings point to a flexible hydrophobic core in the LIM1 domain allowing residual relative mobility of the two zinc fingers, which might be important to optimize the LIM1 interface for interaction with its physiological target molecule(s) and to compensate enthalpically for the entropy loss upon binding.     

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.     

Structure of cysteine- and glycine-rich protein CRP2. Backbone dynamics reveal motional freedom and independent spatial orientation of the lim domains

Members of the cysteine- and glycine-rich protein family (CRP1, CRP2, and CRP3) contain two zinc-binding LIM domains, LIM1 (amino-terminal) and LIM2 (carboxyl-terminal), and are implicated in diverse cellular processes linked to differentiation, growth control, and pathogenesis. Here we report the solution structure of full-length recombinant quail CRP2 as determined by multi-dimensional triple-resonance NMR spectroscopy. The structural analysis revealed that the global fold of the two LIM domains in the context of the full-length protein is identical to the recently determined solution structures of the isolated individual LIM domains of quail CRP2. There is no preference in relative spatial orientation of the two domains. This supports the view that the two LIM domains are independent structural and presumably functional modules of CRP proteins. This is also reflected by the dynamic properties of CRP2 probed by 15N relaxation values (T1, T2, and nuclear Overhauser effect). A model-free analysis revealed local variations in mobility along the backbone of the two LIM domains in the native protein, similar to those observed for the isolated domains. Interestingly, fast and slow motions observed in the 58-amino acid linker region between the two LIM domains endow extensive motional freedom to CRP2. The dynamic analysis indicates independent backbone mobility of the two LIM domains and rules out correlated LIM domain motion in full-length CRP2. The finding that the LIM domains in a protein encompassing multiple LIM motifs are structurally and dynamically independent from each other supports the notion that these proteins may function as adaptor molecules arranging two or more protein constituents into a macromolecular complex.     

Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins

Two classes of homeodomain proteins, Hox and Engrailed, have been shown to act in concert with the atypical homeodomain proteins Pbx and extradenticle. We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with Engrailed-2 (En-2). Furthermore, there are similarities between heterodimeric interactions involving the yeast mating type proteins MATa1 and MATalpha2 and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that were previously shown to form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity. Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeodomain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms.     

Desmosomal cadherin binding domains of plakoglobin

Plakoglobin is a major component of both desmosomes and adherens junctions. At these sites it binds to the cytoplasmic domains of cadherin cell-cell adhesion proteins and regulates their adhesive and cytoskeletal binding functions. Plakoglobin also forms distinct cytosolic protein complexes that function in pathways of tumor suppression and cell fate determination. Recent studies in Xenopus suggest that cadherins inhibit the signaling functions of plakoglobin presumably by sequestering this protein at the membrane and depleting its cytosolic pool. To understand the reciprocal regulation between desmosomal cadherins (desmoglein and desmocollin) and plakoglobin, we have sought to identify the binding domains involved in the formation of these protein complexes. Plakoglobin comprises 13 central repeats flanked by amino-terminal and carboxyl-terminal domains. Our results show that repeats 1-4 are involved in binding desmoglein-1. In contrast, the interaction of plakoglobin with desmocollin-1a is sensitive to deletion of either end of the central repeat domain. The binding sites for two adherens junction components, alpha-catenin and classical cadherins, overlap these sites. Competition among these proteins for binding sites on plakoglobin may therefore account for the distinct composition of adherens junctions and desmosomes.