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...
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