Revealing the glutamate switch: Insights into proton translocation in plant H⁺-pyrophosphatase

Membrane-bound pyrophosphatases (M-PPases) are responsible for the hydrolysis of pyrophosphate (PPi), coupled with the pumping of H⁺ and/or Na⁺ across the membrane. In Vigna radiata H⁺-translocating pyrophosphatase (VrH⁺-PPase), proton translocation involves both a "binding change" mechanism, where PPi binding induces proton translocation, and the "Grotthuss-chain" mechanism, which describes proton translocation along the ion gate, hydrophobic gate, and exit channel. However, the dynamic coupling between protonation states and conformational changes in VrH⁺-PPase remains unclear, partly due to the challenges of experimentally capturing transient states during transport. To address this, we employed constant pH molecular dynamics and classical molecular dynamics simulations to elucidate the proton transport mechanism from the ion gate (R242/D294/K742/E301) to the hydrophobic gate (L232/A305/L555/V746). Our simulations reveal that K742 becomes deprotonated upon PPi binding, suggesting its potential role as an internal proton donor. When K742 is deprotonated, E301 penetrates the hydrophobic gate and creates a hydrophilic environment for proton transport. Following PPi hydrolysis, D294 accepts a hydrolysis-generated proton to become protonated, inducing R242 to act as a positive plug that prevents the reprotonation of D294. Meanwhile, the protonation of D294 causes E301 to rebound and close the hydrophobic gate. We propose that E301 acts as a molecular switch, regulating proton transport through the hydrophobic gate. Furthermore, we suggest that the penetration of glutamate is a conserved feature among plant H⁺-PPases, maintaining a consistent hydrophilic environment at the hydrophobic gate in H⁺-PPases. In conclusion, proton translocation in plant H⁺-PPases involves lysine deprotonation in the PPi-bound state and aspartate protonation in the PPi-hydrolyzed states, with the glutamate switch dynamically regulating the opening and closing of the hydrophobic gate.