Pasquo A, Nardi M C, Dimasi N, Tomei L, Steinkühler C, Delmastro P, Tramontano A, De Francesco R
Istituto di Ricerche di Biologia Molecolare (IRBM), P. Angeletti, Pomezia (Rome), Italy.
Fold Des. 1998;3(6):433-41. doi: 10.1016/S1359-0278(98)00060-1.
The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct.
The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor.
We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.
丙型肝炎病毒NS3蛋白的蛋白酶结构域参与病毒多聚蛋白的成熟过程。NS4A蛋白的一个中央疏水结构域作为其蛋白水解活性的辅因子是必需的。最近已解析了单独的蛋白酶结构域以及与NS4A衍生肽复合的三维结构，结果显示蛋白酶的N端与辅因子的C端非常接近。为了研究其辅因子激活该酶的分子基础，并克服与双分子复合物相关的结构和功能研究困难，我们合理设计了一个连接子来连接这两个分子，以形成单一多肽构建体。
构建的工程化构建体产生了一种具有蛋白水解活性的稳定单体蛋白，其活性不依赖于添加代表NS4A蛋白辅因子结构域的合成肽。该蛋白对蛋白质和合成肽底物均有活性。重组NS4A-NS3单链蛋白酶的光谱和动力学分析表明，其特征与与NS4A辅因子复合的分离NS3蛋白酶结构域重叠。
我们在NS3和NS4A多肽链之间设计了一个非常紧密的连接，理由是这将允许形成更稳定的结构。在所有研究的酶学和物理化学性质方面，工程化的单链酶与与其NS4A辅因子复合的NS3蛋白酶没有区别。
