Abstract. Interaction with extraceUular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847-5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393. We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2-terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine-rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression.

Abstract. Zyxin is an 82-kD protein first identified as a component of adhesion plaques and the termini of stress fibers near where they associate with the cytoplasmic face of the adhesive membrane. We report here that zyxin interacts with the actin cross-linking protein a-actinin . Zyxin cosediments with filamentous actin in an a-actinin-dependent manner and an association between zyxin and a-actinin is observed in solution by analytical gel filtration . The specificity of the interaction between zyxin and a-actinin was demonstrated by blot overlay experiments in which 125 I-zyxin recognizes most prominently a-actinin among a complex mixture of proteins extracted from avian smooth muscle . By these blot overlay binding studies, we determined that zyxin interacts with the NH2-terminal

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.

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...

The LIM motif is a cysteine- and histidine-rich sequence that was first identified in proteins involved in control of gene expression and cell differentiation. In order to characterize structural features of the LIM domain, we have carried out biophysical studies on two polypeptides that display LIM domains: the cysteine-rich intestinal protein (CRIP) and a fragment of the cysteine-rich protein (CRP). Bacterial expression vectors were constructed for the intact CRIP molecule and the C-terminal half of CRP, designated LIM2, such that each expressed protein contained a single LIM domain. Both proteins were recovered as soluble, Zn(II)-containing proteins. The metal coordination properties of these two distinct LIM domain proteins were highly similar, suggesting that a common structural architecture may exist in LIM domain proteins. Both proteins exhibit a maximum of two tetrahedrally bound Zn(II) ions per molecule. Electronic spectroscopy of Co(II) complexes and 113Cd NMR of Cd(II) complexes of CRIP and LIM2 revealed a similar ligand field pattern with one tetrathiolate (S4) site and one S3N1 site for divalent metal ions. The nitrogen ligand was shown to arise from a histidyl imidazole by heteronuclear multiple quantum coherence NMR. The eight conserved residues within the LIM domains of CRIP and LIM2 include seven cysteines and one histidine. It is likely that these conserved residues generate the S4 and S3N1 Zn(II)-binding sites. Metal binding to the two sites within a single LIM domain is sequential, with preferential occupancy of the S4 site. Slow metal ion exchange occurs between sites within an LIM domain, and metal exchange with exogenous metal ions is observed, with exchange at the S3N1 site being kinetically more facile. In the absence of metal binding both proteins appear to be substantially unfolded. Metal binding stabilizes a tertiary fold containing appreciable secondary structural elements. The common metal ion coordination in CRIP and LIM2 suggests that the LIM motif may constitute a structural module with conserved features.