Ekblad C M S, Wilkinson H R, Schymkowitz J W H, Rousseau F, Freund S M V, Itzhaki L S
MRC Centre for Protein Engineering, University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, UK.
J Mol Biol. 2002 Jul 12;320(3):431-42. doi: 10.1016/s0022-2836(02)00478-3.
The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.
乳腺癌易感基因产物BRCA1是一种肿瘤抑制因子，但其在致癌过程中发挥作用的生化和生物学功能仍有待确定。在此，我们对BRCA1高度保守的C末端的溶液性质进行了表征，该末端由BRCT结构域的串联重复序列（BRCT-tan）组成，在BRCA1介导的肿瘤抑制中起关键作用。BRCT-tan的整体解折叠自由能很高（在20℃水中为14.2千卡/摩尔），但其解折叠是通过易于聚集的部分折叠中间体进行的。构建了一组具有代表性的癌症相关序列变体，并测量了它们对蛋白质稳定性的影响。所有突变都具有高度的去稳定性，因此预计会导致功能丧失。超过一半的突变体无法以可溶形式纯化，这表明这些残基对于维持结构完整性至关重要。其余突变体比野生型表现出更大的聚集倾向，这很可能是由于它们相对于部分折叠中间体的热力学稳定性降低所致。此处表征的突变位于BRCT-tan结构的不同位点，这并不能完全解释它们对蛋白质稳定性的影响。因此，结果表明生物物理研究在评估序列变体的重要性以及确定它们如何导致疾病方面具有重要作用。
