Sequential metal binding by the RING finger domain of BRCA1

被引:50
作者
Roehm, PC [1 ]
Berg, JM [1 ]
机构
[1] JOHNS HOPKINS UNIV,SCH MED,DEPT BIOPHYS & BIOPHYS CHEM,BALTIMORE,MD 21205
关键词
D O I
10.1021/bi970863d
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Analysis of the amino acid sequence encoded by the familial breast and ovarian cancer susceptibility gene, BRCA1 [Miki et al. (1994) Science 266, 66-71], revealed the presence of an amino-terminal RING finger domain, a zinc binding motif found in a variety of proteins, Previously determined structures of two RING finger peptides from other proteins revealed that each RING finger sequence forms a single domain that includes two interleaved metal binding sites. One is a four-cysteine site comprised of metal binding residues 1, 2, 5, and 6 (in terms of position along the amino acid sequence) (site 1) and I-he other is a three-cysteine, one-histidine site involving metal binding residues 3, 4, 7, and 8 (site 2). We have characterized the metal binding and metal-dependent folding properties of peptides encompassing the BRCA1 RING finger. Using cobalt(II) as a spectroscopic probe, we have found that metal binding is sequential, with site I becoming nearly fully occupied prior to metal binding To site 2. More detailed thermodynamic analysis as well as studies of a variant peptide revealed that metal binding appears to be anticooperative with dissociation constants of 3 x 10(-8) M for site 1, 5 x 10(-7) M for site 2 with site I unoccupied, and 8; x 10(-6) M for site 2 when site 1 is occupied. Circular dichroism spectroscopic studies revealed that the BRCA1 RING finger peptide is somewhat structured at pH 7 in the absence of metal ions, with further structural changes occurring after the metal binding.
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页码:10240 / 10245
页数:6
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