Molecular Biology II, Center of Medical Biotechnology (ZMB), University of Duisburg-Essen, Universitätsstrasse 5, 45141 Essen, Germany.
Institute of Organic Chemistry I, University of Duisburg-Essen, Universitätsstrasse 7, 45141 Essen, Germany.
Biomacromolecules. 2022 Nov 14;23(11):4504-4518. doi: 10.1021/acs.biomac.2c00652. Epub 2022 Oct 6.
Many natural proteins contain flexible loops utilizing well-defined complementary surface regions of their interacting partners and usually undergo major structural rearrangements to allow perfect binding. The molecular recognition of such flexible structures is still highly challenging due to the inherent conformational dynamics. Notably, protein-protein interactions are on the other hand characterized by a multivalent display of complementary binding partners to enhance molecular affinity and specificity. Imitating this natural concept, we here report the rational design of advanced multivalent supramolecular tweezers that allow addressing two lysine and arginine clusters on a flexible protein surface loop. The protease Taspase 1, which is involved in cancer development, carries a basic bipartite nuclear localization signal (NLS) and thus interacts with Importin α, a prerequisite for proteolytic activation. Newly established synthesis routes enabled us to covalently fuse several tweezer molecules into multivalent NLS ligands. The resulting bi- up to pentavalent constructs were then systematically compared in comprehensive biochemical assays. In this series, the stepwise increase in valency was robustly reflected by the ligands' gradually enhanced potency to disrupt the interaction of Taspase 1 with Importin α, correlated with both higher binding affinity and inhibition of proteolytic activity.
许多天然蛋白质包含利用其相互作用伙伴的明确互补表面区域的柔性环，并且通常经历大的结构重排以允许完美结合。由于固有构象动力学，这种柔性结构的分子识别仍然具有很大的挑战性。值得注意的是，另一方面，蛋白质-蛋白质相互作用的特征是通过多价显示互补结合伙伴来增强分子亲和力和特异性。受此启发，我们在这里报告了先进的多价超分子镊子的合理设计，该镊子允许在柔性蛋白质表面环上寻址两个赖氨酸和精氨酸簇。参与癌症发展的蛋白酶 Taspase 1携带碱性双部分核定位信号（NLS），因此与 Importin α相互作用，这是蛋白水解激活的前提。新建立的合成路线使我们能够将几个镊子分子共价融合成多价 NLS 配体。然后在综合生化测定中系统地比较了所得的双价至五价构建体。在该系列中，配体破坏 Taspase 1 与 Importin α相互作用的能力逐渐增强，这与更高的结合亲和力和抑制蛋白水解活性相关，这种逐步增加的价数得到了稳健的反映。
