Molecular Dynamics Simulations on the Escherichia coli Ammonia Channel Protein AmtB:  Mechanism of Ammonia/Ammonium Transport

Abstract

Molecular dynamics (MD) simulations have been performed at the atomic level to study the ammonium/ammonia transport across the Escherichia coli AmtB membrane protein. Although ammonia primarily exists in the form of NH(4)(+) in aqueous solution, the recent X-ray structure determination of AmtB reveals that the ammonium/ammonia transporter proteins are ammonia-conducting channels rather than ammonium ion transporters [Khademi, S.; et al. Science 2004, 305, 1587; Zheng, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17090]. Our simulations showed that the entrance of NH(4)(+) into the periplasmic recruitment vestibule requires only 3.1 kcal/mol of energy. This is consistent with the X-ray crystal structure, where one NH(4)(+) is captured in the binding vestibule. In this vestibule, NH(4)(+) loses one water of hydration, but the loss is compensated by a hydrogen bond, first with the backbone carbonyl oxygen of Phe161 then with the hydroxyl group of Ser219, as well as the stabilizing pi-cation interactions with the aromatic rings of Trp148 and Phe107 in the AmtB protein. In the end of this recruitment vestibule, the phenyl ring of Phe107 dynamically switches to an open state. This is correlated with a slight rotation and shifting of the indole ring of Trp148, which eventually creates a slot for the initially buried carboxylate group of Asp160 to become exposed to the bulk solvent. A hydrogen bond wire between NH(4)(+) and the carboxylate group of Asp160 via two water molecules was observed. Thus, Asp160 is most likely the proton acceptor from NH(4)(+). This explains the high conservation of Asp160 in Amt proteins and why the D160A mutant would completely quench the activity of AmtB [Javelle, A.; et al. J. Biol. Chem. 2004, 279, 8530; Marini, A. M.; et al. Curr. Genet. 2006, 49, 364]. Once NH(4)(+) deprotonates, the phenyl ring of Phe215 rotates to open, and the subsequent passage of NH(3) through the channel is straightforward.

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

“…Several computational studies suggest that deprotonation takes place in the Phe gate region, but they differ in detail and in the nature of the suggested base accepting the proton (12,13,(15)(16)(17). In accordance with mechanism S these calculations all discuss deprotonation with the view that the released proton will not transit the pore.…”

“…These studies have addressed the strength and specificity of the periplasmic binding site, the nature of ammonium deprotonation and ammonia reprotonation in the vestibule regions, the dynamics and possible function of the highly conserved Phe gate, NH 4 ϩ versus NH 3 conduction through the channel pore, and channel hydration (12)(13)(14)(15)(16)(17)(18)(19). Overall, they agree in predicting strong binding of an ammonium ion at S1, in supporting the view of a highly dynamic Phe gate, and, very importantly, in rejecting the possibility of NH 4 ϩ conduction through the pore because of a high energy barrier.…”

Simulations of anion transport through OprP reveal the molecular basis for high affinity and selectivity for phosphate

“…Free energy profiles (PMFs) have been calculated for a number of permeant species through biological pores, including, for example, K ϩ ions through the KcsA channel (14), water and glycerol through aquaporins and aquaglyceroporins (15)(16)(17), and ammonia through AmtB (21,22). It is therefore useful to compare these pores with OprP.…”

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…Conflicting suggestions with van Heeswijk et al respect to the involvement of the aspartate residue (D160) in deprotonation of ammonium have been uttered. Some argue for a direct involvement (293,294), while others suggest no involvement at all (279,285). Also, the twin histidines have been considered to play a role in deprotonation (290).…”

“…Also, the twin histidines have been considered to play a role in deprotonation (290). Nevertheless, the site most frequently mentioned in this respect is the phenylalanine gate (278,279,284,285,(294)(295)(296)(297). Thus, currently, a preference for the phenylalanine gate as the site of deprotonation seems to develop.…”