Mutational analysis of the metal sites in an LIM domain.
Site-directed mutagenesis was carried out to map the residues that form 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 histidyl 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 S3N1 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 mutagenesis was employed to generate three mutant LIM2 proteins with conversions of the second conserved cysteine to histidine (C2H), the fifth conserved cysteine to histidine (C5H), and the last conserved cysteine to aspartate (C8D). Metal coordination by the mutant proteins was evaluated by atomic absorption spectroscopy, Co(II) electronic spectroscopy, and 113Cd NMR spectroscopy. The results permit discrimination between various models of metal ion binding and suggest that the LIM domain is comprised of a S3N1 site generated from the four N-terminal candidate ligands (CX2CX17HX2C) and a S4 site generated from the four C-terminal candidate ligands (CX2CX17CX2C).
Michelsen
J W
JW
Department of Biology, University of Utah, Salt Lake City 84132.
Sewell
A K
AK
Louis
H A
HA
Olsen
J I
JI
Davis
D R
DR
Winge
D R
DR
Beckerle
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001717:Birds; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D015032:Zinc / Q000378:metabolism*
D002352:Carrier Proteins; D008670:Metals; D010455:Peptides; C017800:zinc-binding protein; D003035:Cobalt; D015032:Zinc