posted on 2024-02-22, 12:37authored byYongLe He, Jinnette Tolentino Collado, James N. Iuliano, Helena A. Woroniecka, Christopher R. Hall, Agnieszka A. Gil, Sergey P. Laptenok, Gregory M. Greetham, Boris Illarionov, Adelbert Bacher, Markus Fischer, Jarrod B. French, Andras Lukacs, Stephen R. Meech, Peter J. Tonge
The blue-light photoreceptor
YtvA from Bacillus
subtilis has an N-terminal flavin mononucleotide (FMN)-binding
light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate
transporter and anti-σ factor antagonist (STAS) output domain.
To interrogate the signal transduction pathway that leads to photoactivation,
the STAS domain was replaced with a histidine kinase, so that photoexcitation
of the flavin could be directly correlated with biological activity.
N94, a conserved Asn that is hydrogen bonded to the FMN C2O
group, was replaced with Ala, Asp, and Ser residues to explore the
role of this residue in triggering the structural dynamics that activate
the output domain. Femtosecond to millisecond time-resolved multiple
probe spectroscopy coupled with a fluorescence polarization assay
revealed that the loss of the hydrogen bond between N94 and the C2O
group decoupled changes in the protein structure from photoexcitation.
In addition, alterations in N94 also decreased the stability of the
Cys-FMN adduct formed in the light-activated state by up to a factor
of ∼25. Collectively, these studies shed light on the role
of the hydrogen bonding network in the LOV β-scaffold in signal
transduction.