Table 2:

Relative affinities for hydronium/sodium binding in the three binding sites of hHv1, estimated from free energy perturbation (FEP) calculations. The total free energy difference was estimated using the following relationship : $\Delta G_{H_3{O}^{+}\to N{a}^{+}=}\Delta G_{H_3{O}^{+}\to {Na}^{+}}^{app}-\Delta G_{H_3{O}^{+}\to N{a}^{+}}^{wat}+\Delta G_{H_3{O}^{+}}^{cst}-\Delta G_{N{a}^{+}}^{cst}$ where ∆G_app is the apparent relative affinity for hydronium over sodium in the binding site as calculated by FEP calculations, ∆G_wat is the reference free energy difference in water, and the two last terms compensate for the free energy loss from enforcing distance restraints in the FEP calculations.

[kcal/mol]	${{\text{ΔG}}_{\text{H3O+>Na+}}}^{\text{app}}$	${{\text{ΔG}}_{\text{H3O+>Na+}}}^{\text{wat}}$	${{\text{ΔG}}_{\text{H30+}}}^{\text{cst}}$	${{\text{ΔG}}_{\text{Na+}}}^{\text{cst}}$	${\text{ΔG}}_{\text{H30+>Na+}}$
Ext	8.1±0.6	3.6±0.4	0.9±1.0	1.5±1.0	4.0±2.0
Core	14.0±0.6	3.6±0.4	1.1±1.0	2.7±1.0	8.8±2.0
Int	8.5±0.6	3.6±0.4	1.2±1.0	2.0±1.0	4.1±2.0