Nassal M, Rieger A, Steinau O
Zentrum für Molekulare Biologie Universität Heidelberg, F.R.G.
J Mol Biol. 1992 Jun 20;225(4):1013-25. doi: 10.1016/0022-2836(92)90101-o.
The nucleocapsid, or core particle, of hepatitis B virus is formed by 180 subunits of the core protein, which contains Cys at positions 48, 61, 107 and 183, the latter constituting the C terminus. Upon adventitious oxidation, some or all of these cysteine residues participate in the formation of disulphide bridges, leading to polymerization of the subunits within the particle. To utilize the cysteine residues as topological probes, we reduced the number of possible intersubunit crosslinks by replacing these residues individually, or in all combinations, by serine. A corresponding set of variants was constructed within the context of an assembly-competent core protein variant that lacks the highly basic C-terminal region. Analysis, by polyacrylamide gel electrophoresis under non-reducing conditions, of the oxidative crosslinking products formed by the wild-type and mutant proteins expressed in Escherichia coli, revealed a clear distinction between the three N-proximal, and the C-terminal Cys: N-proximal Cys formed intermolecular disulphide bonds only with other N-proximal cysteine residues, leading to dimerization. Cys48 and Cys61, in contrast to Cys107, could be crosslinked to the homologous cysteine residues in a second subunit, and are therefore located at the dimer interface. Cys 183 predominantly formed disulphide bonds with Cys183 in subunits other than those crosslinked by the N-proximal cysteine residues. Hence, the polymers generated by oxidation of the wild-type protein are S-S-linked dimeric N-terminal domains interconnected via Cys183/Cys183 disulphide bonds. The intermolecular crosslinks between the N-proximal cysteine residues were apparently the same in the C-terminally truncated and in the full-length proteins, corroborating the model in which the N-terminal domain and the C terminus of the HBV core protein form two distinct and structurally independent entities. The strong tendency of the N-terminal domain for dimeric interactions suggests that core protein dimers are the major intermediates in hepatitis B virus nucleocapsid assembly.
乙型肝炎病毒的核衣壳，即核心颗粒，由核心蛋白的180个亚基组成，该核心蛋白在第48、61、107和183位含有半胱氨酸，后者构成C末端。在偶然氧化时，这些半胱氨酸残基中的一些或全部参与二硫键的形成，导致颗粒内亚基的聚合。为了将半胱氨酸残基用作拓扑探针，我们通过将这些残基单独或以所有组合形式替换为丝氨酸，减少了可能的亚基间交联数量。在一个缺乏高度碱性C末端区域的具有装配能力的核心蛋白变体的背景下构建了相应的一组变体。通过在非还原条件下的聚丙烯酰胺凝胶电泳分析在大肠杆菌中表达的野生型和突变蛋白形成的氧化交联产物，揭示了三个N近端半胱氨酸与C末端半胱氨酸之间的明显区别：N近端半胱氨酸仅与其他N近端半胱氨酸残基形成分子间二硫键，导致二聚化。与Cys107相反，Cys48和Cys61可以与第二个亚基中的同源半胱氨酸残基交联，因此位于二聚体界面。Cys183主要与除了由N近端半胱氨酸残基交联的那些亚基之外的其他亚基中的Cys183形成二硫键。因此，野生型蛋白氧化产生的聚合物是通过Cys183/Cys183二硫键相互连接的S-S连接的二聚体N末端结构域。N近端半胱氨酸残基之间的分子间交联在C末端截短的蛋白和全长蛋白中显然是相同的，这证实了乙型肝炎病毒核心蛋白的N末端结构域和C末端形成两个不同且结构独立的实体的模型。N末端结构域强烈倾向于二聚体相互作用表明核心蛋白二聚体是乙型肝炎病毒核衣壳装配中的主要中间体。
