Mechanistic insights into the aggregation pathway of an immunoglobulin light chain protein

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain protein. In addition, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes prior to organ malfunction and amyloid formation. The structure of these oligomers and the mechanism of toxicity is, however, not understood. In this work, we focus on the patient derived λ-III light chain variable domain FOR005. This protein is mutated at five positions with respect to the closest germline protein GL. In addition, we investigate the single point mutant FOR005-R49G and GL. Using solution-state NMR spectroscopy, we were able to follow the individual steps involved in protein misfolding from the native state to the amyloid fibril structure at atomic resolution for the first time. We show that unfavorable mutations in the complementary determining regions introduce a strain in the protein structure which induces partial unfolding of the native state. Subsequently, the protein converts into a high molecular weight, oligomeric, molten globule like structure. We demonstrate that this transition is driven by electrostatic interactions. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into amyloid fibrils. We find that the topology of the aggregated state is determined by balanced electrostatic interactions in the core of the fibril, resulting in an anti-parallel arrangement of the beta-sheets around the conserved disulfide bond.

系统性抗体轻链（AL）淀粉样变性的特征为：源自特定抗体轻链蛋白的淀粉样原纤维发生沉积。此外，在器官功能障碍与淀粉样蛋白形成前，可溶性寡聚化AL蛋白可直接对心肌细胞产生细胞毒性作用。然而，此类寡聚体的结构与毒性作用机制目前仍不明晰。本研究聚焦于患者来源的λ-III型轻链可变结构域FOR005。相较于与其序列最接近的种系蛋白GL，该蛋白存在5个位点的突变。同时，我们对单点突变体FOR005-R49G以及种系蛋白GL展开研究。本研究首次借助溶液态核磁共振波谱法（solution-state NMR spectroscopy），在原子分辨率下追踪了蛋白质从天然状态到淀粉样原纤维结构的错误折叠全过程。研究表明，互补决定区（complementary determining regions）内的有害突变会在蛋白质结构中引入张力，进而诱导天然状态发生部分解折叠。随后，该蛋白会转变为高分子量、寡聚化的熔球态（molten globule）类似结构。本研究证实，该转变过程由静电相互作用驱动。寡聚体内部易聚集区域的高局部浓度最终会催化其向淀粉样原纤维转化。研究发现，聚集态的拓扑结构由淀粉样原纤维核心区域的平衡静电相互作用所决定，最终使得保守二硫键周围的β折叠片层（beta-sheets）呈现反平行排布。