The Cancer-predisposing Mutation C61G Disrupts Homodimer Formation in the NH2-terminal BRCA1 RING Finger Domain

Peter S Brzovic,Jose Meza,Mary-Claire King,Rachel E Klevit

doi:10.1074/jbc.273.14.7795

Peter S Brzovic, Jose Meza + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.14.7795

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Abstract

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.

Highlights

The BRCA1 tumor suppressor gene encodes for a large protein of 1863 amino acid residues that bears little homology to previously described proteins [1]
The PML and immediate-early EHV-1 protein from equine herpesvirus (IEEHV) solution structures show that the RING finger domain alone can behave as an autonomous Zn2ϩ-dependent folding motif, but the RING finger of RAG1, in combination with a second RAG1 Zn2ϩ-binding module, forms a larger structural unit that homodimerizes in solution
We engineered three constructs of BRCA1, all of which start at the NH2 terminus of BRCA1 and include the RING finger motif

Summary

Introduction

The BRCA1 tumor suppressor gene encodes for a large protein of 1863 amino acid residues that bears little homology to previously described proteins [1]. Another RING finger-containing protein, BARD1 (BRCA1associated RING domain 1), has been identified that interacts with the NH2-terminal region of BRCA1 [9]. The structures share common folding topologies for the core of the domain (Fig. 1), which include at least two antiparallel ␤-strands and tetrahedral coordination of two Zn2ϩ atoms by the eight conserved residues that define the RING finger motif.

Results

Conclusion