Single-molecule reaction chambers reveal intramolecular protein kinase activation
- 1Max Perutz Labs
Abstract
Many protein kinases, ubiquitous signal transducers across all kingdoms of life, are activated by phosphorylation of one or more conserved residues in their activation loops. This reaction is widely considered to be intermolecular (trans) in nature, dependent on the transient dimerization of two kinase domains. However, since the substrate, product and reaction chemistry are identical, bulk solution kinase assays cannot distinguish between intra- and inter-molecular reactions. Discriminating between these two mechanisms is crucial because they impose different requirements on the cell for acute, high-fidelity signal transduction. Using single-molecule reaction chambers fabricated from DNA origami, we show that the paradigmatic insulin receptor kinase (IRK) undergoes intramolecular (cis) autophosphorylation with kinetics indistinguishable from those in free solution. Furthermore, we demonstrate that the bacterial serine/threonine kinase protein kinase B (PknB) undergoes intramolecular autophosphorylation, allosterically stimulated by intermolecular (trans) interactions. Our findings establish that intramolecular activation loop autophosphorylation is not only possible, but likely an ancient and conserved feature of protein kinase signaling. More broadly, our work provides new tools for single-molecule enzymology.
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BibTeX
@misc{kroetenheerdt2026singlemolecule,
title = {{Single-molecule reaction chambers reveal intramolecular protein kinase activation}},
author = {E. Kroetenheerdt and A. Ehrl and C. Stelmach and F. Reina and L. Piech and F. Praetorius and J. A. Ries and B. Zagrovic and T. A. Leonard},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.08.757607},
url = {https://www.biorxiv.org/content/10.64898/2026.10.08.757607v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Single-molecule reaction chambers reveal intramolecular protein kinase activation
AU - E. Kroetenheerdt
AU - A. Ehrl
AU - C. Stelmach
AU - F. Reina
AU - L. Piech
AU - F. Praetorius
AU - J. A. Ries
AU - B. Zagrovic
AU - T. A. Leonard
PY - 2026
DA - 2026/10/08
PB - bioRxiv
DO - 10.64898/2026.10.08.757607
UR - https://www.biorxiv.org/content/10.64898/2026.10.08.757607v1
AB - Many protein kinases, ubiquitous signal transducers across all kingdoms of life, are activated by phosphorylation of one or more conserved residues in their activation loops. This reaction is widely considered to be intermolecular (trans) in nature, dependent on the transient dimerization of two kinase domains. However, since the substrate, product and reaction chemistry are identical, bulk solution kinase assays cannot distinguish between intra- and inter-molecular reactions. Discriminating between these two mechanisms is crucial because they impose different requirements on the cell for acute, high-fidelity signal transduction. Using single-molecule reaction chambers fabricated from DNA origami, we show that the paradigmatic insulin receptor kinase (IRK) undergoes intramolecular (cis) autophosphorylation with kinetics indistinguishable from those in free solution. Furthermore, we demonstrate that the bacterial serine/threonine kinase protein kinase B (PknB) undergoes intramolecular autophosphorylation, allosterically stimulated by intermolecular (trans) interactions. Our findings establish that intramolecular activation loop autophosphorylation is not only possible, but likely an ancient and conserved feature of protein kinase signaling. More broadly, our work provides new tools for single-molecule enzymology.
ER -