Wu Guan-Lin, Shi Yi-Jun, Chiou Jing-Ting, Huang Chia-Hui, Lee Yuan-Chin, Wang Liang-Jun, Chang Long-Sen
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan.
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan; Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.
Arch Biochem Biophys. 2022 Jun 15;722:109209. doi: 10.1016/j.abb.2022.109209. Epub 2022 Apr 1.
In this study, we investigated the functional roles of Asp40, Asp57, and C-terminal Asn60 in Naja atra cardiotoxin 3 (CTX3) structure and function by modifying these three carboxyl groups with semicarbazide. The conjugation of the carboxyl groups with semicarbazide produced two conformational isomers whose gross and fine structures were different from those of CTX3. The blocking of the carboxyl groups increased the structural flexibility of CTX3 in response to trifluoroethanol-induced effect. Despite presenting modest to no effect on decreasing the induction of permeability in zwitterionic phospholipid vesicles, the carboxyl group-modified CTX3 showed a marked reduction in its permeabilizing effect on anionic phospholipid vesicles in comparison to that of the native protein. Compared with native CTX3, carboxyl group-modified CTX3 exhibited lower activity in inducing membrane leakage in U937 cells. The CD spectra of lipid-bound toxins and the color transition of polydiacetylene/lipid assay showed that the membrane interaction mode of CTX3 was distinctly changed by the modification in the carboxyl groups. Given that the selective modification of Asp40 does not cause the conformational isomerization of CTX3, our data indicate that the carboxyl groups in Asp57 and Asn60 are essential in maintaining the structural topology of CTX3. Furthermore, modification of carboxyl groups changes the interdependence between the infrastructure and the global conformation of CTX3 in modulating membrane permeabilizing activity.
在本研究中，我们通过用氨基脲修饰这三个羧基，研究了眼镜蛇毒心脏毒素3（CTX3）中Asp40、Asp57和C末端Asn60在其结构和功能中的作用。羧基与氨基脲的结合产生了两种构象异构体，其总体和精细结构与CTX3不同。羧基的封闭增加了CTX3在三氟乙醇诱导作用下的结构灵活性。尽管对两性离子磷脂囊泡中通透性诱导的降低作用不大或没有作用，但与天然蛋白相比，羧基修饰的CTX3对阴离子磷脂囊泡的通透作用明显降低。与天然CTX3相比，羧基修饰的CTX3在诱导U937细胞的膜泄漏方面表现出较低的活性。脂质结合毒素的圆二色光谱和聚二乙炔/脂质测定的颜色转变表明，CTX3的膜相互作用模式因羧基修饰而明显改变。鉴于对Asp40的选择性修饰不会导致CTX3的构象异构化，我们的数据表明，Asp57和Asn60中的羧基对于维持CTX3的结构拓扑至关重要。此外，羧基的修饰改变了CTX3在调节膜通透活性时基础结构与整体构象之间的相互依存关系。
