TY - JOUR
T1 - Elucidating the signal transduction mechanism of the blue-light-regulated photoreceptor YtvA: From photoactivation to downstream regulation
AU - He, YongLe
AU - Collado, Jinnette Tolentino
AU - Iuliano, James N.
AU - Woroniecka, Helena A.
AU - Hall, Christopher R.
AU - Gil, Agnieszka A.
AU - Laptenok, Sergey P.
AU - Greetham, Gregory M.
AU - Illarionov, Boris
AU - Bacher, Adelbert
AU - Fischer, Markus
AU - French, Jarrod B.
AU - Lukacs, Andras
AU - Meech, Stephen R.
AU - Tonge, Peter J.
N1 - Funding Information: J.T.C. was supported by the National Institutes of Health IMSD-MERGE (T32GM135746) and NY-CAPs IRACDA (K12-GM102778) Programs at Stony Brook University. A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557) and was supported by PTE ÁOK-KA-2021. This study was supported by the National Science Foundation (NSF) (MCB-1817837 to P.J.T.) and the EPSRC (EP/N033647/1 to S.R.M.). Y.H. and J.N.I were supported by a National Institutes of Health Chemistry-Biology Interface Training Grant (T32GM092714). The authors are grateful to STFC for access to the ULTRA laser facility.
PY - 2024/3/15
Y1 - 2024/3/15
N2 - 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 C2═O 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 C2═O 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.
AB - 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 C2═O 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 C2═O 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.
UR - https://www.scopus.com/pages/publications/85186242806
U2 - 10.1021/acschembio.3c00722
DO - 10.1021/acschembio.3c00722
M3 - Article
SN - 1554-8929
VL - 19
SP - 696
EP - 706
JO - ACS Chemical Biology
JF - ACS Chemical Biology
IS - 3
ER -