Neutron diffraction study to reveal the novel cryoprotectant mechanism of para-hydroxy benzoic acid and its binding to HosA transcription factor by potential hydronium ions

We propose a neutron diffraction study to investigate the role of hydronium ions in the novel cryoprotectant properties of para-hydroxy benzoic acid (PHB), a sparingly water-soluble organic molecule, observed in X-ray crystallography of protein crystals. PHB exhibits remarkable cryoprotective effects in a proprietary water-based solution with negligible amounts of other solvents, suggesting a hydronium-driven mechanism forming water shell networks. Co-crystal X-ray structures of PHB with a pathogenic bacterial transcription factor HosA, supported by molecular dynamics, revealed a water molecule bound to PHB and carried between sites during simulation. After PHB binding to the exact site on HosA, the water molecule, likely a hydronium ion, interacts with four partners, including a repulsive interaction with a positively charged residue. Diffraction studies at I23 (DLS) detected no anomalous signal at the water site, ruling out a metal ion at this position. Simultaneous neutron diffraction of PHB crystals and HosA-PHB co-crystals can confirm the presence of hydronium ions and provide insight into the possible water shell network orchestrated by them. This discovery could enhance cryoprotection strategies in structural biology improving crystal stability for diffraction successes. Moreover, understanding the possible hydronium-based binding mechanism of PHB to HosA, a key protein in bacterial motility, may expedite drug discovery efforts.