Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210, USA.
Structural Biology and Bio-Informatics Division, Indian Institute of Chemical Biology, Kolkata, 700032, India.
Sci Rep. 2017 Sep 4;7(1):10339. doi: 10.1038/s41598-017-10906-w.
The conformational dynamics of a pathogenic κ4 human immunoglobulin light-chain variable domain, SMA, associated with AL amyloidosis, were investigated by N relaxation dispersion NMR spectroscopy. Compared to a homologous light-chain, LEN, which differs from SMA at eight positions but is non-amyloidogenic in vivo, we find that multiple residues in SMA clustered around the N-terminus and CDR loops experience considerable conformational exchange broadening caused by millisecond timescale protein motions, consistent with a destabilized dimer interface. To evaluate the contribution of each amino acid substitution to shaping the dynamic conformational landscape of SMA, NMR studies were performed for each SMA-like point mutant of LEN followed by in silico analysis for a subset of these proteins. These studies show that a combination of only three mutations located within or directly adjacent to CDR3 loop at the dimer interface, which remarkably include both destabilizing (Q89H and Y96Q) and stabilizing (T94H) mutations, largely accounts for the differences in conformational flexibility between LEN and SMA. Collectively, our studies indicate that a correct combination of stabilizing and destabilizing mutations is key for immunoglobulin light-chains populating unfolded intermediates that result in amyloid formation, and underscore the complex nature of correlations between light-chain conformational flexibility, thermodynamic stability and amyloidogenicity.
通过 N 弛豫分散 NMR 光谱研究了与 AL 淀粉样变性相关的致病性 κ4 人免疫球蛋白轻链可变域 SMA 的构象动力学。与 SMA 仅在 8 个位置不同但在体内无淀粉样变性的同源轻链 LEN 相比，我们发现 SMA 中多个位于 N 端和 CDR 环附近的残基经历了由毫秒时间尺度蛋白质运动引起的相当大的构象交换展宽，这与不稳定的二聚体界面一致。为了评估每个氨基酸取代对塑造 SMA 动态构象景观的贡献，对每个 SMA 样 LEN 点突变进行了 NMR 研究，然后对其中一些蛋白质进行了计算分析。这些研究表明，仅在二聚体界面内或直接紧邻 CDR3 环的三个突变的组合，其中包括显著的不稳定（Q89H 和 Y96Q）和稳定（T94H）突变，在很大程度上解释了 LEN 和 SMA 之间构象灵活性的差异。总的来说，我们的研究表明，正确组合的稳定和不稳定突变是免疫球蛋白轻链占据导致淀粉样形成的无折叠中间体的关键，并且强调了轻链构象灵活性、热力学稳定性和淀粉样变性之间相关性的复杂性质。
