The LIM region of a presumptive Caenorhabditis elegans transcription factor is an iron-sulfur- and zinc-containing metallodomain.

Abstract

The cysteine-rich LIM motif is highly conserved between invertebrates and mammals. This motif shows similarit both to proteins that bind zinc and to ferredoxins, which contain iron-sulfur dusters. Two tandem copies of the LIM motif are found in a number of presumptive transcription factors, including the protein product of the Caenorhabdits ekgans cell-lineage gene lH-11. To investigate the possible metal-binding properties of the LIM region of the lin-li protein, we expressed and purified a 151-amino acid peptide containing the tandem LIM motifs. The purified peptide binds both zinc (two atoms per protein molecule) and iron (as a redox-active iron-sulfur cluster, with four atoms of iron and four atoms of inorganic sulfide per protein molecule). These observations suggest that the LIM motif is a metaflodomain that might function in a redox-sensitive regulation of transcription.The cysteine-rich motif referred to as LIM shows similarity to the metal-binding domains of both zinc metalloproteins and ferredoxins (1-3). The LIM motif, Cys-Xaa2-CysXaa17.19-His-Xaa2-Cys-Xaa2-Cys-Xaa2-Cys-Xaa7nll-(Cys)-Xaa8-Cys, is present in two tandem repeats in a number of proteins found in invertebrates and mammals ( Fig. 1 a and b). This conservation among highly diverged species suggests that the LIM region corresponds to a functionally important protein domain. The LIM motif was first identified in the protein products of three genes ( Fig. 1): lin-l, which controls certain asymmetric cell divisions during vulval development of Caenorhabditis elegans (1); Isl-1, which encodes an insulin gene enhancer-binding protein from rat (2); and mec-3, which is required for the differentiation of certain C. elegans mechanosensory neurons (4). The acronym LIM (1, 2) derives from these three gene names: jin-hl, Isl-1, and mec-3. All three genes encode proteins that contain two tandem copies of the LIM motif and also a homeodomain, which has been implicated in DNA binding and transcriptional regulation (5). Two tandem copies ofthe LIM motifare also encoded by two genes that do not encode a homeodomain: Ttg-1 or rhombotin (6, 7), which is located at the breakpoint of a chromosomal translocation in a T-cell leukemia, and a gene similar to Ttg-1 in sequence, Rhom-2 (3).The function of the LIM region is not known. The conserved cysteine and histidine residues in the LIM motif have been proposed to be responsible for the ligation of metal ions (1, 2). To investigate the possible metal-binding properties of the LIM region, we expressed the LIM region of lin-il as a 151-amino acid fragment in Escherichia coli. Upon purification, this LIM polypeptide was found to contain four atoms of iron in the form of iron-sulfur cluster(s) and two atoms of zinc per protein molecule. MATERIALS AND METHODSConstruction of Expression Plasmids. DNA corresponding to the LIM region of the lin-li gene was amplified using the expression-cassette polymerase chain reaction (PCR) method (8). Two synthetic oligonucleotide primers were used. Primer 1 (5'-GGGCCGCTAGAAGGAG...

Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton.

“…It has been postulated that LIM domains might interact with either nucleic acids or protein (see for example Freyd et al, 1990;Boehm et al, 1990a;Li et al, 1991). We suggest that LIM domains serve as protein-binding interfaces since, as discussed below, we have demonstrated a direct interaction between zyxin and a protein that is comprised primarily of LIM domains.…”

“…We have shown here that zyxin isolated from avian smooth muscle binds zinc, but exhibits no detectable iron, cadmium or copper. In contrast with the metal profile of zyxin, bacterially expressed LIM sequences derived from Lin-ll have been reported to bind zinc as well as iron in a redox sensitive iron-sulfur cluster (Li et al, 1991). The reason for the apparent difference in metal liganding by zyxin and Lin-11 has not been resolved.…”

Molecular cloning and functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adaptor.

“…4A). In both of these regions, the arrangment of these two amino acids does not reveal any similarity to other known Cys/ His-rich motifs, for example, the LIM domain (Li et al 1991) or the RING motif (Freemont et al 1991). Overall, p300 is rich in prolines, glutamines, and serines, which together constitute >30% of all amino acid residues of the protein.…”

Redox control of transcription: sensors, response regulators, activators and repressers

“…However, there is no obvious reason why redox control of gene expression should be restricted to prokaryotic cells. The lin-11 gene product of Caenorhabditis elegans [30] is probably a redox activator protein as defined here. The product of the n$S gene of yeast chromosome III [31] may also belong to this class.…”

“…The haem-binding domain exists as a module that is found in other sensor proteins [18] The E. coli redox response regulator, ArcA, has 238 amino acids and is phosphorylated by ArcB on . The N-terminal domain is likely to have a structure similar to that of CheY, a bacterial chemotaxis response Caenorhabditis elegans [30] regulator the structure of which has been determined by NMR spectroscopy [20,21]. CheY is phosphorylated on Asp-57, which is located on a surface-exposed loop between the first two anti-parallel /?-strands.…”