MicroID: A Novel Biotin Ligase Enables Rapid Proximity Ligation Proteomics

Identifying protein-protein interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity ligation method that uses a promiscuous biotin ligase to detect protein-protein interactions in cells in a highly reproducible manner. Recent advancements in proximity ligation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2 to create a smaller, highly active, biotin ligase that we named MicroID. Truncation of the c-terminal of BioID2 and mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller, highly active construct with lower baseline labeling. Several additional point mutations improved the function of our modified MicroID construct compared to BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID is the smallest biotin ligase (180 AA for microID vs. 257 AA for miniTurbo and 338 AA for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurboID. MicroID also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we developed a MicroID mutant that has lower labeling efficiency, but significantly reduced biotin scavenging compared to BioID2. Finally, we demonstrate utility of MicroID in mass spectrometry experiments by localizing MicroID constructs to subcellular organelles and measuring protein-protein interactions.

鉴定蛋白质-蛋白质相互作用，是解析细胞内信号传导与调控通路的核心环节。BioID是一类邻近连接技术，借助广谱生物素连接酶，可通过高度可重复的方式检测细胞内的蛋白质-蛋白质相互作用。近年来，邻近连接工具的技术革新提升了标记效率并优化了标记时序，使得研究人员能够在细胞生理时间尺度上实现蛋白质相互作用的检测。然而，这类重组构建体仍存在分子量偏大、稳定性不足以及背景标记水平过高等问题。本研究通过改造BioID2的结构，开发出一种体积更小、活性更高的生物素连接酶，并将其命名为MicroID。通过截短BioID2的C端（C-terminal），并引入突变以解除生物素/ATP在BioID2活性位点的结合阻滞，我们获得了体积更小、活性更高且本底标记水平更低的重组构建体。额外引入的若干点突变进一步优化了改造后的MicroID构建体的功能，其性能优于BioID2以及TurboID、miniTurbo等其他生物素连接酶。MicroID是目前体积最小的生物素连接酶（MicroID仅含180个氨基酸（Amino Acid, AA），对比miniTurbo的257个AA、TurboID的338个AA），其标记活性仅略低于TurboID，且性能优于miniTurboID。同时，MicroID的背景标记水平也低于TurboID。针对需要对标记过程实现精确时序调控的实验场景，我们开发了一款MicroID突变体：其标记效率有所降低，但与BioID2相比，生物素清除能力得到了显著改善。最后，我们通过将MicroID构建体靶向定位至亚细胞细胞器，并以此检测蛋白质-蛋白质相互作用，验证了MicroID在质谱实验中的应用潜力。