The LIM motif defines a specific zinc-binding protein domain.

Abstract

The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-ll, Isl-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are remiiniscent of a metalbinding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) The LIM motif is a cysteine-rich sequence found in a diverse collection of proteins including transcription factors (1-5), a protooncogene product (6, 7), and cytoskeletal components (8-11). Many of the LIM proteins appear to be involved in regulation of gene expression and cellular differentiation during development. The specific function of the LIM domain has not been established, although it has been postulated to serve as a DNA or protein binding interface. Because of the abundance of conserved cysteine residues in the LIM consensus sequence, the motif has been widely proposed to be a metal-binding sequence. Efforts have been made to examine the metal-binding properties of LIM-motif proteins (12,13). For example, it has been demonstrated that the cysteine-rich intestinal protein (CRIP) will bind exogenously applied Zn ions (13). In addition, expression of the LIM domains of Lin-1l in Escherichia coli resulted in the isolation of a protein from inclusion bodies that contained both Zn and an Fe-S cluster (12). The observation of an Fe-S cluster in Lin-1l prompted consideration of the intriguing idea that the LIM transcription factors might be redox-regulated (12).In this report we present a comprehensive metal analysis of a LIM-domain protein isolated from its endogenous source. Specifically, we describe the metal-binding properties of the chicken cysteine-rich protein (cCRP) isolated from avian smooth muscle (10). cCRP, the chicken homologue of the human CRP (11), exhibits two LIM domains of the sequence CX2CX17HX2CX2CX2CX17CX2C (J. Pino and M.C.B., unpublished results). Our results show cCRP to be a Zn(II) metalloprotein. The implications of these results for the LIM domain structure are discussed. MATERIALS AND METHODSPurification of cCRP. cCRP was purified from fresh chicken gizzards by a procedure to be described in detail elsewhere (A. W. Crawford, J. D. Pino, and M.C.B., unpublished work). The purity of cCRP was demonstrated by SDS/PAGE and amino acid analysis. An extinction coefficient for cCRP of 2.66 x 104 M-1'cm-' was obtained by measurement of the absorbance at 280 nm followed by quantitation of the cCRP protein by amino acid analysis. Thiol titrations were carried out as described (14, 15).Metal Exchange. cCRP was prepared for metal exchange reactions by dialysis in buffer M (40 mM Tris Cl, pH 7.5/40 mM KCl). The protein was diluted 10-fold with 0.2 M potassium phosphate (pH 7.2) and subsequently incubated with mentioned quantities of metal salts. Spectra were recorded after 10 min. For measurements of binding stoichiometry, the metal-replaced samples were incubated for 1-3 hr, dialyzed in buffer M containi...

FHL2 Regulates Cell Cycle-Dependent and Doxorubicin-Induced p21Cip1/Waf1 Expression in Breast Cancer Cells

“…One intact LIM domain contains two zinc fingers which mediate protein-protein interactions. 2,3 Due to the presence of four and a half LIM domains, members of the FHL subfamily are able to interact with a broad variety of proteins. There are five members described so far, FHL1-FHL4 and ACT (activator of CREM in testis).…”

Isolation, tissue expression, and chromosomal assignment of a human LIM protein gene, showing homology to rat Enigma homologue (ENH)

“…LIM domain proteins have been implicated in developmental regulation, cellular differentiation, and actin-based cytoskeletal interaction (Sadler et al 1992;Crawford et al 1994;Sanchez-Garcia and Rabbitts 1994). Spectroscopic studies of LIM domains derived from a number of different proteins have revealed that the LIM domain specifically coordinates two zinc ions (Michelsen et al 1993Archer et al 1994;Kosa et al 1994). Although many zinc finger motifs are involved in DNA or RNA binding (Klug and Schwabe 1995), the LIM domain has been proposed to participate in protein-protein interactions Dawid et al 1995Dawid et al , 1998.…”

PINCH-1 Is an Obligate Partner of Integrin-linked Kinase (ILK) Functioning in Cell Shape Modulation, Motility, and Survival

“…PINCH is a family of proteins consisting of a tandem array of five LIM domains, structures that are involved in mediating protein-protein interactions (8,9). We have previously found that PINCH-1, the founding member of the PINCH family (10), is a cytoplasmic component of cell-ECM adhesions (11,12).…”