39AT

Crystal structure of LUCI-tag, ligand-free

Summary for 39AT

• Entry DOI: 10.2210/pdb39at/pdb
• Descriptor: LUCI-tag, self-labeling protein, GUANOSINE-5'-DIPHOSPHATE, MAGNESIUM ION, ... (4 entities in total)
• Functional Keywords: k-ras(g12c), sequence redesign, gtpase, self-labeling protein, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 2
• Total formula weight: 38261.45

Authors

Mou, J. (deposition date: 2026-09-27, release date: 2026-10-28)

Primary citation

Mou, J.,Leong, J.W.,Gao, S.,Slaw, K.,McGill, M.,Donovan, K.A.,Metivier, R.J.,Sendker, F.L.,Kong, Q.,Lin, D.,Dharani, A.M.,Chen, X.D.,Haigis, K.M.,Fischer, E.S.,Chen, F.,Polizzi, N.F.
Deep-learning-based design of an orthogonal self-labeling protein from K-Ras(G12C).
Biorxiv, 2026

PubMed Abstract: 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.

PubMed: 42856208
DOI: 10.64898/2026.09.27.754867

Experimental method

X-RAY DIFFRACTION (2.08 Å)