Mechanism of voltage gating in the voltage-sensing phosphatase Ci-VSP.

Conformational changes in voltage-sensing domains (VSDs) are driven by the transmembrane electric field acting on the protein charges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes as well as the major conformations visited during a complete functional gating cycle in the isolated VSD of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1 to S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated "Up" and resting "Down" states, the simulations predict two Ci-VSD conformations: a deeper resting state ("down-minus") and an extended activated ("up-plus") state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. The present results show that these four states are visited sequentially in a stepwise manner during voltage activation, each step translocating one arginine or the equivalent of ∼1 e 0 across the membrane electric field, yielding a transfer of ∼3 e 0 charges in total for the complete process.