Paramyxovirus fusion protein: characterization of the core trimer, a rod-shaped complex with helices in anti-parallel orientation.
Paramyxovirus fusion protein: characterization of the core trimer, a rod-shaped complex with helices in anti-parallel orientation.
复制标题
副粘病毒融合蛋白：核心三聚体的表征，核心三聚体是一种具有反平行方向螺旋的杆状复合物。
作者：
Dutch,RE;Leser,GP;Lamb,RA
副粘病毒SV5的融合(F)蛋白含有两个七肽重复区域，HRA邻近融合肽，HRB邻近跨膜结构域。分别对应于这些七肽重复区域的肽 N-1 和 C-1 形成异二聚体的热稳定 α-螺旋三聚体（S. B. Joshi、R. E. Dutch 和 R. A. Lamb (1998).Virology 248,20–34）。 N-1/C-1 复合物的进一步表征表明，预计残留在复合物外部的 C-1 肽当存在于复合物中时能够抵抗多种蛋白酶的消化。只有蛋白酶 K 消化了大部分 C-1 肽，尽管 C-1 剩余的少量蛋白酶保护片段赋予了蛋白酶 K 抗性 N-1 三聚卷曲螺旋极高的热稳定性。 N-1/C-1 复合物的羧肽酶 Y 消化表明 C-1 肽相对于 N-1 肽以反平行方向缔合。 N-1/C-1复合物的电镜显示为杆状复合物，平均长度为9.7 nm，与N-1均以α螺旋形式存在一致。通过圆二色性测量分析，发现 N-1 的七肽重复序列和残基（预计向内指向 N-1 三聚体卷曲螺旋中心的位置）的突变具有不同的影响。突变 I137M 不影响分离的 N-1 肽的螺旋结构，但影响 N-1/C-1 复合物的热稳定性。突变 L140M 和 L161M 扰乱了 N-1 单独形成的螺旋结构，但不影响耐热 N-1/C-1 复合物的形成。最后，分析了对应于所提出的亮氨酸拉链区域的肽 SV5 F 255-293 对 N-1、C-1 或 N-1/C-1 复合物的影响。圆二色性分析表明，虽然肽 255-293 的存在增加了来自 N-1 或 N-1/C-1 复合物的螺旋信号，但没有观察到热稳定性的变化，表明该区域不是 F 蛋白最终、最稳定核心的组成部分。
The fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, α-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998).Virology 248,20–34). Further characterization of the N-1/C-1 complex indicated that the C-1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an α helix. Mutations at heptad repeataanddresidues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as analyzed by circular dichroism measurements. The mutation I137M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L140M and L161M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein.