The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Victoria 3010, Australia; Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia.
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Victoria 3010, Australia.
Arch Biochem Biophys. 2014 Mar 1;545:33-43. doi: 10.1016/j.abb.2014.01.003. Epub 2014 Jan 13.
Activation and oligomerisation of Bax, a key pro-apoptotic Bcl-2 family protein, are key steps in the mitochondrial pathway to apoptosis. The signals for apoptosis are conveyed by the distantly related BH3-only proteins, which use their short BH3 domain, an amphipathic α-helix, to interact with other Bcl-2 family members. Here we report an NMR study of interactions between BaxΔC and BH3 domain-containing peptides in the absence and presence of CHAPS, a zwitterionic detergent. We find for the first time that CHAPS interacts weakly with BaxΔC (fast exchange on the NMR chemical shift timescale), at concentrations below micelle formation and with an estimated Kd in the tens of mM. Direct and relatively strong-interactions (slow exchange on the NMR chemical shift timescale) were also observed for BaxΔC with BaxBH3 (estimated Kd of circa 150μM) or BimBH3 in the absence of CHAPS. The interaction with either peptide alone induced widespread chemical shift perturbations to BaxΔC in solution which implies that BaxΔC might have undergone significant conformation change upon binding the BH3 peptide. However, BaxΔC remained monomeric upon binding either CHAPS or a BH3 peptide alone, but the presence of both provoked it to form a dimer.
Bax 的激活和寡聚化，一种关键的促凋亡 Bcl-2 家族蛋白，是细胞凋亡线粒体途径中的关键步骤。凋亡信号是由远亲 BH3-only 蛋白传递的，它们利用其短的 BH3 结构域，一个两亲性的α螺旋，与其他 Bcl-2 家族成员相互作用。在这里，我们报告了一项关于 BaxΔC 与 BH3 结构域含有肽段在无 CHAPS 和存在 CHAPS 情况下相互作用的 NMR 研究。我们首次发现 CHAPS 与 BaxΔC 弱相互作用（在 NMR 化学位移时间尺度上快速交换），在低于胶束形成的浓度下，估计 Kd 在数十 mM 左右。在没有 CHAPS 的情况下，BaxΔC 与 BaxBH3（估计 Kd 约为 150μM）或 BimBH3 也存在直接和相对较强的相互作用（在 NMR 化学位移时间尺度上缓慢交换）。与单个肽段的相互作用会导致 BaxΔC 在溶液中发生广泛的化学位移扰动，这意味着 BaxΔC 可能在结合 BH3 肽后发生了显著的构象变化。然而，BaxΔC 在单独结合 CHAPS 或 BH3 肽时仍保持单体状态，但两者的存在会促使其形成二聚体。
