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.     

Suppression in transformed avian fibroblasts of a gene (crp) encoding a cysteine-rich protein containing LIM domains

Using cDNA subtraction and differential hybridization techniques, a cDNA library derived from normal quail embryo fibroblasts was screened for clones corresponding to genes whose expression was suppressed in v-myc-transformed, as compared with normal, quail embryo fibroblasts. One of the isolated cDNA clones corresponded to a 0.9-kb mRNA that was present in normal quail and chicken embryo fibroblasts, but was virtually absent from all transformed avian cells tested: quail embryo fibroblasts transformed by the v-myc, v-myc/v-mil or v-src oncogenes, cells derived from a methylcholanthrene-induced quail fibrosarcoma or v-myc-transformed chicken macrophages. Nucleotide sequence analysis of the original and supplementary cDNA clones indicated that the corresponding gene encodes a 194 amino acid cysteine-rich protein (M(r) 20,911). A database search revealed that the gene is the avian homolog of a human primary response gene (crp) of unknown function. Both the quail and human CRP proteins contain two copies of a cysteine-rich amino acid sequence motif (LIM) with putative zinc-binding activity that was previously identified in several proteins with presumed regulatory functions essential for cell growth or differentiation.     

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.     

The LIM motif defines a specific zinc-binding protein domain

The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.     

Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.     

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.     

Molecular cloning and characterization of the filarial LIM domain proteins AvL3-1 and OvL3-1

A full-length cDNA of the filarial nematode Acanthocheilonema viteae was isolated from a cDNA library of female worms, using a partial cDNA of the OvL3-1 gene of Onchocerca volvulus as a probe. The AvL3-1 cDNA contained an open reading frame which encoded for a protein with a theoretical molecular weight of 64 kDa. The deduced protein contained a predicted signal sequence, a short repetitive motive of unknown function, and three LIM domains. The structure of the LIM domains was identical to those of zyxin, a cytoskeleton-associated protein of chicken fibroblasts, suggesting that AvL3-1 has a similar role in filarial nematodes. The sequence information was used to isolate the homologous cDNA of O. volvulus by PCR from a cDNA library of female O. volvulus, which showed an overall identity of 76.9% to AvL3-1 on the protein level. AvL3-1 was expressed in Escherichia coli and the affinity-purified fusion free protein was used to immunized jirds (Meriones unguiculatus). Immunization together with the adjuvant STP or with Freund's adjuvant induced IgG and IgM antibody responses, but no significant protection against a challenge infection with L3 of A. viteae, compared to appropriate control groups.     

Structure of the carboxy-terminal LIM domain from the cysteine rich protein CRP

The three dimensional solution structure of the carboxy terminal LIM domain of the avian Cysteine Rich Protein (CRP) has been determined by nuclear magnetic resonance spectroscopy. The domain contains two zinc atoms bound independently in CCHC (C = Cys, H = His) and CCCC modules. Both modules contain two orthogonally-arranged antiparallel beta-sheets, and the CCCC module contains an alpha-helix at its C terminus. The modules pack due to hydrophobic interactions forming a novel global fold. The structure of the C-terminal CCCC module is essentially identical to that observed for the DNA-interactive CCCC modules of the GATA-1 and steroid hormone receptor DNA binding domains, raising the possibility that the LIM motif may have a DNA binding function.     

Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.     

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.