THE LIM MOTIF DEFINES A SPECIFIC ZINC-BINDING PROTEIN DOMAIN

The cysteine-rich protein (CRP) contains two copies of the LIM sequenc e Motif, CX2CX17HX2CX2CX2CX17CX2C, that was first identified in the ho meodomain proteins Lin-11, Isl-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal- binding domain. We examined the metal-binding properties of CRP isolat ed from chicken smooth muscle (cCRP) and from a bacterial expression s ystem and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic s tudies of Co(II)- and Cd-113(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dom inating the ligand types. One metal site within each LIM motif has tet rathiolate (S4) coordination, the second site may either be S4 or S3N1 . The LIM motif represents another example of a specific Zn-binding pr otein sequence.