Chemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools such as the FK506-binding protein–FKBP–rapamycin-binding– (FKBP–FRB)–rapamycin system have been widely used to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) does not exist, due to inherent challenges in designing a chemical that simultaneously binds three proteins with high affinity and specificity. Here, we developed CIT by rationally splitting FRB and FKBP. Cellular and structural datasets showed efficient trimerization of split pairs of FRB or FKBP with full-length FKBP or FRB, respectively, by rapamycin. CIT rapidly induced tri-organellar junctions and perturbed intended membrane lipids exclusively at select membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells to address cell biology questions otherwise intractable and engineer cell functions for future synthetic biology applications.

化学诱导二聚化 (CID) 使用小分子诱导两种不同蛋白质的结合。FK506 结合蛋白-FKBP-雷帕​​霉素结合-(FKBP-FRB)-雷帕霉素系统等 CID 工具已被广泛用于探测细胞内外的分子事件。虽然有各种 CID 工具可用，但化学诱导三聚化 (CIT) 并不存在，因为在设计一种同时以高亲和力和特异性结合三种蛋白质的化学物质时存在固有的挑战。在这里，我们通过合理拆分 FRB 和 FKBP 来开发 CIT。细胞和结构数据集显示 FRB 或 FKBP 的分裂对与全长 FKBP 或 FRB 分别被雷帕霉素有效三聚化。CIT 迅速诱导三细胞器连接，并专门在选定的膜接触部位扰乱预期的膜脂。