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Biochemistry. 1997 Aug 19;36(33):10240-5.

Sequential metal binding by the RING finger domain of BRCA1.

Author information

1
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.

Abstract

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 the 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 1 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 1 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.

PMID:
9254622
DOI:
10.1021/bi970863d
[Indexed for MEDLINE]

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