Substitution of aspartic acid with glutamic acid at position 67 of the BRCA1 RING domain retains ubiquitin ligase activity and zinc(II) binding with a reduced transition temperature.
Breast cancer susceptibility protein 1 (BRCA1) participates in genomic integrity maintenance through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain which forms two Zn(2+) binding sites in an interleaved fashion. A number of deleterious BRCA1 missense mutations, which predispose an individual to a subset of hereditary breast and ovarian cancers, have been identified in the RING domain. Disruption of Zn(2+) binding sites and protein structure results in the inactivation of BRCA1 tumor suppression function. An unprecedented D67E BRCA1 mutation, identified in Thai familial breast cancer patients, is located in the vicinity of Zn(2+) binding site II, and its pathogenic significance remains elusive. The present study revealed that the D67E BRCA1 RING protein assumes a preformed structure in the absence of Zn(2+). The Zn(2+)-bound mutant protein was more folded, resulting in enhanced proteolytic resistance and dimerization. This indicated that the mutation retained Zn(2+) binding, and barely perturbed the native global structure of the BRCA1 RING domain. The complex between D67E BRCA1 and BARD1 RING domains exhibited a substantial ubiquitin ligase activity compared with a defective complex containing the C61G BRCA1 mutation. However, the D67E mutation was slightly less stable toward thermal denaturation. This implies that the D67E mutation might be a neutral or mild cancer-risk modifier of other defective mechanisms underlying BRCA1-mutation-related breast cancer.
Laboratory of Pharmaceutical Biotechnology, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla, 90112, Thailand.
Apichart
A
Atipairin
Bhutorn
B
Canyuk
Adisorn
A
Ratanaphan
D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D019313:BRCA1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005260:Female; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D056928:X-Ray Absorption Spectroscopy; D015032:Zinc / Q000378:metabolism
D019313:BRCA1 Protein; D001224:Aspartic Acid; D018698:Glutamic Acid; D044767:Ubiquitin-Protein Ligases; D015032:Zinc