Zhang Huiqin, Liu Yong, Li Yunyan, Ruan Maosen, Zhou Shu, Wang Junfeng, Yang Jing
Institute of Physical Science and Information Technology, Anhui University, 230061, Hefei, China.
High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China.
Magn Reson Lett. 2024 Jun 27;5(1):200141. doi: 10.1016/j.mrl.2024.200141. eCollection 2025 Feb.
HCLS1-associated protein X-1 (HAX1) is a multifunctional mitochondrial protein involved in the regulation of apoptosis, a crucial process of programmed cell death, and mRNA processing. Despite its significance, limited structural data is available for HAX1, hindering a comprehensive understanding of its biological function. Notably, the caseinolytic mitochondrial matrix peptidase chaperone subunit B (CLPB) has been identified as an interacting partner of HAX1, yet the biophysical properties and binding affinity governing their interaction remain poorly defined. In this study, we present a thorough biophysical characterization of full-length human HAX1 and CLPB, accomplished through recombinant expression and purification. By employing size exclusion chromatography, dynamic light scattering, and circular dichroism spectroscopy, we successfully established their biophysical properties, revealing contrasting structural features, with CLPB displaying α-helical content and HAX1 exhibiting a disordered nature. Moreover, we employed solution-state nuclear magnetic resonance (NMR) spectroscopy to probe their binding affinity. Our findings demonstrate the formation of stable multimeric complexes between HAX1 and CLPB, and we quantified a dissociation constant in the low range of micro-molar for their high affinity interaction. These results lay the foundation for further in-depth investigations into the dynamics and energetics governing the HAX1-CLPB interaction, ultimately contributing to a comprehensive understanding of their functional mechanisms.
造血细胞特异性 Lyn 底物 1 相关蛋白 X-1（HAX1）是一种多功能线粒体蛋白，参与细胞凋亡（程序性细胞死亡的关键过程）的调控以及 mRNA 加工。尽管其具有重要意义，但关于 HAX1 的结构数据有限，这阻碍了对其生物学功能的全面理解。值得注意的是，酪蛋白水解线粒体基质肽酶伴侣亚基 B（CLPB）已被确定为 HAX1 的相互作用伙伴，然而，支配它们相互作用的生物物理特性和结合亲和力仍不清楚。在本研究中，我们通过重组表达和纯化对全长人 HAX1 和 CLPB 进行了全面的生物物理表征。通过使用尺寸排阻色谱、动态光散射和圆二色光谱，我们成功确定了它们的生物物理特性，揭示了截然不同的结构特征，CLPB 呈现出α-螺旋结构，而 HAX1 表现出无序性质。此外，我们采用溶液态核磁共振（NMR）光谱来探测它们的结合亲和力。我们的研究结果表明 HAX1 和 CLPB 之间形成了稳定的多聚体复合物，并且我们对它们高亲和力相互作用的解离常数进行了定量，其处于低微摩尔范围内。这些结果为进一步深入研究 HAX1-CLPB 相互作用的动力学和能量学奠定了基础，最终有助于全面理解它们的功能机制。
