A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation.
A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation.
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DOI：
10.1021/jp403425z
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发表时间：
2013-10-24
期刊：
影响因子：
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通讯作者：
Dumoulin M
中科院分区：
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作者：
De Genst E;Chan PH;Pardon E;Hsu SD;Kumita JR;Christodoulou J;Menzer L;Chirgadze DY;Robinson CV;Muyldermans S;Matagne A;Wyns L;Dobson CM;Dumoulin M
我们报告了两种骆驼抗体片段（通常称为纳米抗体，即cAb-HuL 5和通过蛋白质工程获得的稳定且更抗聚集的变体cAb-HuL 5G）的相互作用对人溶菌酶的两种淀粉样蛋白生成变体I56 T和D 67 H的性质的影响，所述淀粉样蛋白生成变体I56 T和D 67 H在重要器官（包括肝、肾和脾）中的沉积与家族性非神经性系统性淀粉样变性相关。核磁共振光谱和X射线晶体学研究均表明，cAb-HuL 5与α结构域结合，该结构域是天然溶菌酶结构的两个叶之一。cAb-HuL 5/cAb-HuL 5G的结合强烈抑制淀粉样蛋白生成变体的原纤维形成;然而，它不抑制这些变体的特征性局部瞬时协同解折叠转变，其中β结构域和C螺旋解折叠，并且其代表淀粉样蛋白原纤维形成中的关键早期中间物质。因此，与先前描述的两种其他纳米抗体不同，cAb-HuL 5/cAb-HuL 5G不通过将溶菌酶变体的天然结构的全局协同性恢复为野生型蛋白质的特征来抑制原纤维形成。相反，它抑制了原纤维组装的后续步骤，包括α结构域的解折叠和结构重组。这些结果表明，纳米抗体可以防止在蛋白质从可溶性天然状态到淀粉样蛋白原纤维的结构转变的不同阶段形成致病性聚集体，说明它们作为结构探针研究淀粉样蛋白原纤维形成的分子机制的价值。结合它们对蛋白质工程技术的适应性以改善其稳定性和溶解性，这些发现支持了纳米抗体可以潜在地开发为治疗蛋白质错误折叠疾病的建议。
We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the α-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the β-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the α-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases.