Louis John M, Ishima Rieko, Nesheiwat Issa, Pannell Lewis K, Lynch Shannon M, Torchia Dennis A, Gronenborn Angela M
Laboratory of Chemical Physics, National Institute Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2003 Feb 21;278(8):6085-92. doi: 10.1074/jbc.M209726200. Epub 2002 Dec 4.
Interactions between the C-terminal interface residues (96-99) of the mature HIV-1 protease were shown to be essential for dimerization, whereas the N-terminal residues () and Arg(87) contribute to dimer stability (Ishima, R., Ghirlando, R., Tozser, J., Gronenborn, A. M., Torchia, D. A., and Louis, J. M. (2001) J. Biol. Chem. 276, 49110-49116). Here we show that the intramonomer interaction between the side chains of Asp(29) and Arg(87) influences dimerization significantly more than the intermonomer interaction between Asp(29) and Arg(8'). Several mutants, including T26A, destablize the dimer, exhibit a monomer fold, and are prone to aggregation. To alleviate this undesirable property, we designed proteins in which the N- and C-terminal regions can be linked intramolecularly by disulfide bonds. In particular, cysteine residues were introduced at positions 2 and 97 or 98. A procedure for the efficient preparation of intrachain-linked polypeptides is presented, and it is demonstrated that the Q2C/L97C variant exhibits a native-like single subunit fold. It is anticipated that monomeric proteases of this kind will aid in the discovery of novel inhibitors aimed at binding to the monomer at the dimerization interface. This extends the target area of current inhibitors, all of which bind across the active site formed by both subunits in the active dimer.
成熟HIV-1蛋白酶C末端界面残基（96 - 99）之间的相互作用被证明对二聚化至关重要，而N末端残基（）和精氨酸（87）则有助于二聚体的稳定性（Ishima, R., Ghirlando, R., Tozser, J., Gronenborn, A. M., Torchia, D. A., and Louis, J. M. (2001) J. Biol. Chem. 276, 49110 - 49116）。在这里我们表明，天冬氨酸（29）和精氨酸（87）侧链之间的单体内部相互作用对二聚化的影响远大于天冬氨酸（29）和精氨酸（8'）之间的单体间相互作用。包括T26A在内的几个突变体使二聚体不稳定，呈现单体构象，并且易于聚集。为了减轻这种不良特性，我们设计了可以通过二硫键在分子内连接N末端和C末端区域的蛋白质。特别是，在位置2和97或98引入了半胱氨酸残基。本文介绍了一种高效制备链内连接多肽的方法，并证明Q2C/L97C变体呈现出类似天然的单亚基构象。预计这类单体蛋白酶将有助于发现旨在在二聚化界面与单体结合的新型抑制剂。这扩展了当前抑制剂的作用靶点范围，目前所有抑制剂都是结合在活性二聚体中由两个亚基形成的活性位点上。
