Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions

Bing Yang, Shibing Tang, Cheng Ma, Shang-Tong Li, Guang-Can Shao, Bobo Dang, William F. DeGrado, Meng-Qiu Dong, Peng George Wang, Sheng Ding and Lei Wang ()
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Bing Yang: University of California San Francisco
Shibing Tang: University of California San Francisco
Cheng Ma: Georgia State University
Shang-Tong Li: National Institute of Biological Sciences
Guang-Can Shao: National Institute of Biological Sciences
Bobo Dang: University of California San Francisco
William F. DeGrado: University of California San Francisco
Meng-Qiu Dong: National Institute of Biological Sciences
Peng George Wang: Georgia State University
Sheng Ding: University of California San Francisco
Lei Wang: University of California San Francisco

Nature Communications, 2017, vol. 8, issue 1, 1-10

Abstract: Abstract Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells. Obviating an external trigger for reactivity and affording residue specificity, GECX enables the capture of low-affinity protein binding (affibody with Z protein), elusive enzyme-substrate interaction (ubiquitin-conjugating enzyme UBE2D3 with substrate PCNA), and endogenous proteins interacting with thioredoxin in E. coli cells, allowing for mass spectrometric identification of interacting proteins and crosslinking sites. This live cell chemistry-based approach should be valuable for investigating currently intangible protein interactions in vivo for better understanding of biology in physiological settings.

Date: 2017
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DOI: 10.1038/s41467-017-02409-z

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