Deep-learning-based design of an orthogonal self-labeling protein from K-Ras(G12C)

Self-labeling protein (SLP) tags enable versatile labeling of proteins in live cells, yet only two SLPs are commonly used, limiting multiplexing. Here, we repurposed the oncoprotein K-Ras(G12C) and its covalent inhibitors to create a third, orthogonal SLP system. We used a deep-learning-based approach to radically redesign the sequence of K-Ras(G12C) while preserving its covalent-inhibitor binding pocket. Our top design, LUCI-tag, is a 19 kDa, monomeric, thermostable SLP that rapidly and covalently reacts with commercially available K-Ras(G12C) inhibitors bearing diverse payloads. Unlike existing SLPs, LUCI-tag exhibited payload-agnostic labeling kinetics, outperforming HaloTag7 and SNAP-tag for a negatively charged payload. X-ray crystal structures of drug-bound and drug-free LUCI-tag showed the design was structurally accurate and contained a preorganized inhibitor-binding pocket that could explain its rapid labeling kinetics. Proteomics and cell-signaling experiments confirmed LUCI-tag is biologically inert. LUCI-tag enabled rapid, wash-free, live-cell imaging and simultaneous three-color multiplexed experiments with HaloTag7 and SNAP-tag. This work establishes LUCI-tag as an immediately useful orthogonal SLP and demonstrates that covalent drug-target pairs can be repurposed into a broadly applicable platform for protein labeling.