Protonation states of ammonia/ammonium in the hydrophobic pore of ammonia transporter protein AmtB.

The crystal structure of the ammonia transport (Amt) protein AmtB at 1.4 Angstrom resolution revealed four ammonia/ammonium (NH(3)/NH(4)(+)) binding sites along the approximately 20 Angstrom narrow pore. It is an open question whether the bound NH(3)/NH(4)(+) are neutral (NH(3)) or cationic (NH(4)(+)). On the basis of the AmtB crystal structure, we calculated the pK(a) of these four NH(3)/NH(4)(+) by solving the Poisson-Boltzmann equation. Except for one NH(3)/NH(4)(+) binding site (Am1) at the entry point of the Amt pore, binding sites are occupied by NH(3) due to lack of energy contributions from solvation, eliminating an existence of charged form NH(4)(+) and, inevitably, its potential cation-pi interaction. The only two titratable residues in the pore, His168 and His318, are in the neutral charge state. The NH(4)(+) charge state at the Am1 site is stabilized by Ser219 functioning as an H-bond acceptor. However, when involving explicit crystal water nearby, the NH(3) charge state is stabilized by the reorientation of Ser219-OH group. This H-bond donor Ser219 significantly decreases the pK(a) of NH(3)/ NH(4)(+) at the Am1 site to approximately 1. The flip/flop H-bond of Ser219 may play a dual role first in binding and subsequently in deprotonating NH(4)(+), which is a prerequisite to conduct NH(3) through the Amt pore across the membrane.