Cornillez-Ty Cromwell T, Lazinski David W
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Virol. 2003 Oct;77(19):10314-26. doi: 10.1128/jvi.77.19.10314-10326.2003.
Hepatitis delta virus expresses two essential proteins, the small and large delta antigens, and both are required for viral propagation. Proper function of each protein depends on the presence of a common amino-terminal multimerization domain. A crystal structure, solved using a peptide fragment that contained residues 12 to 60, depicts the formation of an octameric ring composed of antiparallel coiled-coil dimers. Because this crystal structure was solved for only a fragment of the delta antigens, it is unknown whether octamers actually form in vivo at physiological protein concentrations and in the context of either intact delta antigen. To test the relevance of the octameric structure, we developed a new method to probe coiled-coil structures in vivo. We generated a panel of mutants containing cysteine substitutions at strategic locations within the predicted monomer-monomer interface and the dimer-dimer interface. Since the small delta antigen contains no cysteine residues, treatment of cell extracts with a mild oxidizing reagent was expected to induce disulfide bond formation only when the appropriate pairs of cysteine substitution mutants were coexpressed. We indeed found that, in vivo, both the small and large delta antigens assembled as antiparallel coiled-coil dimers. Likewise, we found that both proteins could assume an octameric quaternary structure in vivo. Finally, during the course of these experiments, we found that unprenylated large delta antigen molecules could be disulfide cross-linked via the sole cysteine residue located within the carboxy terminus. Therefore, in vivo, the C terminus likely provides an additional site of protein-protein interaction for the large delta antigen.
丁型肝炎病毒表达两种必需蛋白，即小δ抗原和大δ抗原，两者都是病毒繁殖所必需的。每种蛋白的正常功能取决于一个共同的氨基末端多聚化结构域的存在。使用包含12至60位残基的肽片段解析得到的晶体结构描绘了由反平行卷曲螺旋二聚体组成的八聚体环的形成。由于该晶体结构仅针对δ抗原的一个片段解析得到，因此尚不清楚八聚体在生理蛋白浓度下以及在完整δ抗原的背景下是否真的在体内形成。为了测试八聚体结构的相关性，我们开发了一种新方法来探测体内的卷曲螺旋结构。我们构建了一组突变体，这些突变体在预测的单体-单体界面和二聚体-二聚体界面的关键位置含有半胱氨酸取代。由于小δ抗原不含半胱氨酸残基，因此预期用温和的氧化试剂处理细胞提取物时，只有当适当的半胱氨酸取代突变体对共表达时才会诱导二硫键形成。我们确实发现，在体内，小δ抗原和大δ抗原均组装成反平行卷曲螺旋二聚体。同样，我们发现这两种蛋白在体内都可以呈现八聚体四级结构。最后，在这些实验过程中，我们发现未异戊烯化的大δ抗原分子可以通过位于羧基末端的唯一半胱氨酸残基进行二硫键交联。因此，在体内，C末端可能为大δ抗原提供了一个额外的蛋白质-蛋白质相互作用位点。
