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Class 1 Sugar Beet Phytoglobin Shows Strong Affinity to Glyceraldehyde-3-Phosphate Dehydrogenase and DNA In Vitro

Groth, Leonard LU ; Oda, Miho LU and Bülow, Leif LU (2025) In International Journal of Molecular Sciences 26(19).
Abstract
Class 1 phytoglobins (Pgbs) are known for their multifunctional roles in
plant stress responses, with recent studies suggesting broader
interactions involving metabolic and transcriptional regulation.
Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
moonlights in many roles in colocalized spaces during cellular stress
that are strikingly suitable for supporting Pgb function. This study
investigates the molecular interactions of class 1 Pgb from sugar beet (Beta vulgaris),
BvPgb 1.2, and an alanine-substituted mutant (C86A), focusing on their
ability to bind GAPDH and DNA. Using dual-emission isothermal spectral
shift (SpS) analysis, we report strong binding interactions with... (More)
Class 1 phytoglobins (Pgbs) are known for their multifunctional roles in
plant stress responses, with recent studies suggesting broader
interactions involving metabolic and transcriptional regulation.
Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
moonlights in many roles in colocalized spaces during cellular stress
that are strikingly suitable for supporting Pgb function. This study
investigates the molecular interactions of class 1 Pgb from sugar beet (Beta vulgaris),
BvPgb 1.2, and an alanine-substituted mutant (C86A), focusing on their
ability to bind GAPDH and DNA. Using dual-emission isothermal spectral
shift (SpS) analysis, we report strong binding interactions with GAPDH,
with dissociation constants (KD) of 260 ± 50 nM for the
recombinant wild-type protein (rWT) and a significantly stronger
affinity for C86A (120 ± 40 nM), suggesting that the cysteine residue
limits the interaction. Remarkably strong DNA-binding affinities were
also observed for both variants, displaying biphasic binding. This
behavior is characteristic of hexacoordinated globins and reflects the
presence of two distinct species: a fast-reacting open pentacoordinated
form and a slow-reacting closed hexacoordinated form with high apparent
affinity. Here, the KD in the open configuration was 120 ± 50
nm and 50 ± 20 nM for rWT and C86A, respectively. In the closed
configuration, however, the cysteine appears to support the interaction,
as the KD was measured at 100 ± 10 pM and 230 ± 60 pM for
rWT and C86A, respectively. Protein–protein docking studies reinforced
these findings, revealing electrostatically driven interactions between
BvPgb 1.2 and GAPDH, characterized by a substantial buried surface area
indicative of a stable, biologically relevant complex. Protein–DNA
docking similarly confirmed energetically favorable binding near the
heme pocket without obstructing ligand accessibility. Together, these
findings indicate a potential regulatory role for BvPgb 1.2 through its
interaction with GAPDH and DNA. (Less)
Please use this url to cite or link to this publication:
author
; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
International Journal of Molecular Sciences
volume
26
issue
19
article number
9404
pages
19 pages
publisher
MDPI AG
ISSN
1422-0067
DOI
10.3390/ijms26199404
language
English
LU publication?
yes
id
7afbddce-c598-42d5-be40-dc8c63ee8b34
date added to LUP
2025-09-26 14:45:04
date last changed
2025-09-29 13:31:47
@article{7afbddce-c598-42d5-be40-dc8c63ee8b34,
  abstract     = {{Class 1 phytoglobins (Pgbs) are known for their multifunctional roles in<br>
 plant stress responses, with recent studies suggesting broader <br>
interactions involving metabolic and transcriptional regulation. <br>
Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) <br>
moonlights in many roles in colocalized spaces during cellular stress <br>
that are strikingly suitable for supporting Pgb function. This study <br>
investigates the molecular interactions of class 1 Pgb from sugar beet (Beta vulgaris),<br>
 BvPgb 1.2, and an alanine-substituted mutant (C86A), focusing on their <br>
ability to bind GAPDH and DNA. Using dual-emission isothermal spectral <br>
shift (SpS) analysis, we report strong binding interactions with GAPDH, <br>
with dissociation constants (K<sub>D</sub>) of 260 ± 50 nM for the <br>
recombinant wild-type protein (rWT) and a significantly stronger <br>
affinity for C86A (120 ± 40 nM), suggesting that the cysteine residue <br>
limits the interaction. Remarkably strong DNA-binding affinities were <br>
also observed for both variants, displaying biphasic binding. This <br>
behavior is characteristic of hexacoordinated globins and reflects the <br>
presence of two distinct species: a fast-reacting open pentacoordinated <br>
form and a slow-reacting closed hexacoordinated form with high apparent <br>
affinity. Here, the K<sub>D</sub> in the open configuration was 120 ± 50<br>
 nm and 50 ± 20 nM for rWT and C86A, respectively. In the closed <br>
configuration, however, the cysteine appears to support the interaction,<br>
 as the K<sub>D</sub> was measured at 100 ± 10 pM and 230 ± 60 pM for <br>
rWT and C86A, respectively. Protein–protein docking studies reinforced <br>
these findings, revealing electrostatically driven interactions between <br>
BvPgb 1.2 and GAPDH, characterized by a substantial buried surface area <br>
indicative of a stable, biologically relevant complex. Protein–DNA <br>
docking similarly confirmed energetically favorable binding near the <br>
heme pocket without obstructing ligand accessibility. Together, these <br>
findings indicate a potential regulatory role for BvPgb 1.2 through its <br>
interaction with GAPDH and DNA.}},
  author       = {{Groth, Leonard and Oda, Miho and Bülow, Leif}},
  issn         = {{1422-0067}},
  language     = {{eng}},
  month        = {{09}},
  number       = {{19}},
  publisher    = {{MDPI AG}},
  series       = {{International Journal of Molecular Sciences}},
  title        = {{Class 1 Sugar Beet Phytoglobin Shows Strong Affinity to Glyceraldehyde-3-Phosphate Dehydrogenase and DNA In Vitro}},
  url          = {{http://dx.doi.org/10.3390/ijms26199404}},
  doi          = {{10.3390/ijms26199404}},
  volume       = {{26}},
  year         = {{2025}},
}