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).