Binding of Monovalent and Bivalent Ligands by Transthyretin Causes Different Short- and Long-Distance Conformational Changes

The wild type protein, transthyretin (TTR), and over 120 genetic TTR variants are amyloidogenic and cause, respectively, sporadic and hereditary systemic TTR amyloidosis. The homotetrameric TTR contains two identical thyroxine binding pockets, occupation of which by specific ligands can inhibit TTR amyloidogenesis in vitro. Ligand binding stabilizes the tetramer, inhibiting its proteolytic cleavage and its dissociation. Here, we show with solution-state NMR that ligand binding induces long-distance conformational changes in the TTR that have not previously been detected by X-ray crystallography, consistently with the inhibition of the cleavage of the DE loop. The NMR findings, coupled with surface plasmon resonance measurements, have identified dynamic exchange processes underlying the negative cooperativity of binding of “monovalent” ligand tafamidis. In contrast, mds84, our prototypic “bivalent” ligand, which is a more potent stabilizer of TTR in vitro that occupies both thyroxine pockets and the intramolecular channel between them, has greater structural effects.野生型转甲状腺素蛋白（transthyretin, TTR）以及超过120种遗传性TTR变异体均具有淀粉样变性潜能，分别引发散发性与遗传性系统性TTR淀粉样变性。同源四聚体TTR包含两个完全相同的甲状腺素结合口袋，特异性配体占据该口袋可在体外抑制TTR的淀粉样生成过程。配体结合可稳定四聚体结构，抑制其蛋白水解切割与解离。本研究通过溶液态核磁共振（solution-state NMR）技术证实，配体结合可诱导TTR发生此前未被X射线晶体学（X-ray crystallography）检测到的长距离构象变化，这与DE环（DE loop）切割的抑制效应相一致。结合表面等离子体共振（surface plasmon resonance）检测结果，该核磁共振研究明确了“单价”配体他法米司特（tafamidis）结合过程中负协同性背后的动态交换机制。与之形成对比的是，我们研发的原型“双价”配体mds84——一种可同时占据两个甲状腺素结合口袋及其间分子内通道的体外强效TTR稳定剂——对TTR的结构影响更为显著。