COMMON METAL-ION COORDINATION IN LIM DOMAIN PROTEINS

The LIM motif is a cysteine- and histidine-rich sequence that was firs t identified in proteins involved in control of gene expression and ce ll differentiation. In order to characterize structural features of th e LIM domain, we have carried out biophysical studies on two polypepti des that display LIM domains: the cysteine-rich intestinal protein (CR IP) and a fragment of the cysteine-rich protein (CRP). Bacterial expre ssion vectors were constructed for the intact CRIP molecule and the C- terminal half of CRP, designated LIM2, such that each expressed protei n contained a single LIM domain. Both proteins were recovered as solub le, Zn(Il)-containing proteins. The metal coordination properties of t hese 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) io ns per molecule. Electronic spectroscopy of Co(II) complexes and Cd-11 3 NMR of Cd(II) complexes of CRIP and LIM2 revealed a similar ligand f ield pattern with one tetrathiolate (S4) site and one S3N1 site for di valent metal ions. The nitrogen ligand was shown to arise from a histi dyl imidazole by heteronuclear multiple quantum coherence NMR. The eig ht conserved residues within the LIM domains of CRIP and LIM2 include seven cysteines and one histidine. It is likely that these conserved r esidues generate the S4 and S3N1 Zn(Il)-binding sites. Metal binding t o the two sites within a single LIM domain is sequential, with prefere ntial occupancy of the S4 site. Slow metal ion exchange occurs between sites within an LIM domain, and metal exchange with exogenous metal i ons is observed, with exchange at the S3N1 site being kinetically more facile. In the absence of metal binding both proteins appear to be su bstantially unfolded. Metal binding stabilizes a tertiary fold contain ing appreciable secondary structural elements. The common metal ion co ordination in CRIP and LIM2 suggests that the LIM motif may constitute a structural module with conserved features.