Roehm P C, Berg J M
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Biochemistry. 1997 Aug 19;36(33):10240-5. doi: 10.1021/bi970863d.
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.
对家族性乳腺癌和卵巢癌易感基因BRCA1[Miki等人（1994年），《科学》266卷，66 - 71页]编码的氨基酸序列进行分析后发现，其氨基末端存在一个RING指结构域，这是一种在多种蛋白质中都能找到的锌结合基序。先前确定的来自其他蛋白质的两种RING指肽段的结构表明，每个RING指序列都形成一个单一结构域，该结构域包含两个交错的金属结合位点。一个是由金属结合残基1、2、5和6（按照氨基酸序列中的位置）组成的四半胱氨酸位点（位点1），另一个是涉及金属结合残基3、4、7和8的三半胱氨酸、一组氨酸位点（位点2）。我们已经对包含BRCA1 RING指结构域的肽段的金属结合和金属依赖性折叠特性进行了表征。使用钴（II）作为光谱探针，我们发现金属结合是顺序性的，在位点2结合金属之前，位点1几乎已被完全占据。更详细的热力学分析以及对一个变体肽段的研究表明，金属结合似乎是反协同的，位点1的解离常数为3×10⁻⁸ M，位点2在未被位点1占据时的解离常数为5×10⁻⁷ M，而当位点1被占据时，位点2的解离常数为8×10⁻⁶ M。圆二色光谱研究表明，BRCA1 RING指肽段在pH 7且无金属离子的情况下具有一定结构，在金属结合后会发生进一步的结构变化。
