Ultrasmall chemogenetic tags with group-transfer ligands

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Chemogenetic tags are essential for studying protein function when small-molecule ligands are unavailable.
• Existing tags are often too large, limiting their application with certain proteins of interest (POIs).

Purpose of the Study:

• To develop and characterize novel, ultrasmall chemogenetic tags for protein research.
• To demonstrate the utility of these tags in cell signaling and proximity induction applications.

Main Methods:

• Engineered two ultrasmall chemogenetic tags, mgTag (36 aa) and cTag (50 aa), with transferase-type reactivity.
• Utilized engineered cysteine residues within the tags for group-transfer reactions with specific ligands.
• Assessed tag functionality by fusing them to KRASG12D and Abelson kinase (ABL), and by inducing proximity between ABL and BRD4.

Main Results:

• mgTag and cTag are the smallest reported chemogenetic tags, enabling functional protein studies without disruption.
• Fusion of mgTag or cTag to KRASG12D did not impair its growth-signaling pathway activation, unlike the larger HaloTag.
• Proximity induction between ABL and BRD4 was achieved using mgTag/cTag and a BRD4 binder, leading to BRD4 phosphorylation.

Conclusions:

• Ultrasmall chemogenetic tags (mgTag, cTag) offer a significant advancement for studying protein function and engineering biological systems.
• The transferase-type reactivity and small size of these tags provide versatile applications in basic science, biotechnology, and medicine.