PROTEIN CHEMISTRY AND STRUCTURE
The Cancer-predisposing Mutation C61G Disrupts Homodimer Formation in the NH2-terminal BRCA1 RING Finger Domain*

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The breast and ovarian cancer tumor suppressor gene, BRCA1, encodes for a Zn2+-binding RING finger motif located near the protein NH2 terminus. The RING finger motif is characterized by eight conserved Cys and His residues which form two Zn2+-binding sites termed Site I and Site II. We used limited proteolysis in conjunction with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy to investigate the metal binding properties and to probe the solution structures of wild-type and mutant BRCA1 constructs that include the RING finger. Our results show that the RING finger motif is part of a larger proteolysis-resistant structural domain which encompasses the first 110 residues of BRCA1. Analytical gel-filtration chromatography and chemical cross-linking experiments demonstrate that the BRCA1 NH2-terminal domain readily homodimerizes in solution. The cancer-predisposing C61G mutation, which alters a conserved Zn2+-binding residue, abolishes metal binding to Site II of the RING finger motif, while Site I remains intact and functional. The C61G mutation also results in increased proteolytic susceptibility of the COOH-terminal portion of the NH2-terminal domain and perturbs the oligomerization properties of BRCA1.

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This work was supported by National Institutes of Health Grant RO1 GM46701 (to R. E. K.) and RO1 CA27632 (to M. C. K.) and by a generous contribution from the Boeing Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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These authors contributed equally to this work.

Supported in part by an American Cancer Society postdoctoral fellowship.