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

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 (<i>Beta vulgaris</i>), 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 (K_D) 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 K_D 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 K_D 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.