1.
Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor.
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Yousefi, OS
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Günther, M
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Hörner, M
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Chalupsky, J
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Wess, M
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Brandl, SM
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Smith, RW
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Fleck, C
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Kunkel, T
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Zurbriggen, MD
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Höfer, T
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Weber, W
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Schamel, WWA
Abstract:
The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.
2.
Interactions Between phyB and PIF Proteins Alter Thermal Reversion Reactions in vitro.
Abstract:
The dynamic behavior of the plant red/far-red light photoreceptor phytochrome B (phyB) has been elucidated in natural and synthetic systems. Red light switches phyB from the inactive Pr state to the active Pfr state, a process that is reversed by far-red light. Alongside light signals, phyB activity is constrained by thermal reversion (that is prominent in the dark) and protein-protein interactions between phyB, other phytochrome molecules, and, among others, PHYTOCHROME INTERACTING FACTORs (PIFs). Requirements for phyB-PIF association have been well studied and are central to light-regulated synthetic tools. However, it is unknown whether PIF interactions influence transitions of phyB between different conformers. Here, we show that the in vitro thermal reversion of phyB involves multiple reactions. Thermal reversion of phyB in vitro is inhibited by PIF6, and this effect is observed at all temperatures tested. We analyzed our experimental data using a mathematical model containing multiple Pfr conformers, in accordance with previous findings. Remarkably, each Pfr conformer is differentially regulated by PIF6 and temperature. As a result, we speculate that in vivo phytochrome signaling networks may require similar levels of complexity to fine-tune responses to the external environment.
