MUTATIONAL ANALYSIS OF THE METAL SITES IN AN LIM DOMAIN

Site-directed mutagenesis was carried out to map the residues that for m the two Zn(II) sites within a LIM domain. The C-terminal LIM domain derived from the cysteine-rich protein was utilized for this analysis and is referred to as LIM2. Seven cysteinyl residues and a single hist idyl residue in the LIM2 sequence, CX2CX17HX2CX2CX2CX17CX2C, comprise the conserved residues in the LIM consensus that are potential Zn(II) ligands. Two Zn(II) binding sites exhibiting tetrathiolate (S4) and SN 3N1 Zn(II) coordination are displayed by LIM2 (Kosa, J. L., Michelsen, J. W., Louis, H. A., Olsen, J. I., Davis, D. R., Beckerle, M. C., and Winge, D. R. (1994) Biochemistry 33, 468-477). Site-directed mutagene sis was employed to generate three mutant LIM2 proteins with conversio ns of the second conserved cysteine to histidine (C2H), the fifth cons erved cysteine to histidine (C5H), and the last conserved cysteine to aspartate (C8D). Metal coordination by the mutant proteins was evaluat ed by atomic absorption spectroscopy, Co(II) electronic spectroscopy, and Cd-113 NMR spectroscopy. The results permit discrimination between various models of metal ion binding and suggest that the LIM domain i s comprised of a S3N1 site generated from the four N-terminal candidat e ligands (CX2CX17HX2C) and a S4 site generated from the four C-termin al candidate ligands (CX2CX17CX2C).