Shihao Wang

Ammonium transporters achieve charge transfer by fragmenting their substrate.

Proteins of the Amt/MEP family facilitate ammonium transport across the membranes of plants, fungi, and bacteria and are essential for growth in nitrogen-poor environments. Some are known to facilitate the diffusion of the neutral NH(3), while others, notably in plants, transport the positively charged NH(4)(+). On the basis of the structural data for AmtB from Escherichia coli , we illustrate the mechanism by which proteins from the Amt family can sustain electrogenic transport. Free energy calculations show that NH(4)(+) is stable in the AmtB pore, reaching a binding site from which it can spontaneously transfer a proton to a pore-lining histidine residue (His168). The substrate diffuses down the pore in the form of NH(3), while the excess proton is cotransported through a highly conserved hydrogen-bonded His168-His318 pair. This constitutes a novel permeation mechanism that confers to the histidine dyad an essential mechanistic role that was so far unknown.

Transport mechanisms in the ammonium transporter family

Ammonium transport is mediated by membrane proteins of the ubiquitous Amt/Rh family. Despite the availability of different X-ray structures that provide many insights on the ammonium permeation process, the molecular details of its mechanism remain controversial. The X-ray structures have revealed that the pore of the Amt and Rh proteins is characterized by a hydrophobic portion about 12A long in which electronic density was observed in crystallographic study of AmtB from Escherichia coli. This electronic density was initially only observed when crystals were grown in presence of ammonium salt and was thus attributed to ammonia (NH(3)) molecules, and lead the authors to suggest that the conduction mechanism in the Amt/Rh proteins involves the single-file diffusion of NH(3) molecules. However, other X-ray crystallography results and molecular mechanics simulations suggest that the pore of AmtB could also be filled with water molecules. The possible presence of water molecules in the pore lumen calls for a reassessment of the growing consensus that Amt/Rh proteins work as plain NH(3) channels. Indeed, functional experiments on plant ammonium transporters and rhesus proteins suggest a variety of permeation mechanisms including the passive diffusion of NH(3), the antiport of NH(4)(+)/H(+), the transport of NH(4)(+), or the cotransport of NH(3)/H(+). We discuss these mechanisms in light of some recent functional and simulation studies on the AmtB transporter and illustrate how they can be reconciled with the available high resolution X-ray data.

Mechanism of NH4+ Recruitment and NH3 Transport in Rh Proteins

In human cells, membrane proteins of the rhesus (Rh) family excrete ammonium and play a role in pH regulation. Based on high-resolution structures, Rh proteins are generally understood to act as NH3 channels. Given that cell membranes are permeable to gases like NH3, the role of such proteins remains a paradox. Using molecular and quantum mechanical calculations, we show that a crystallographically identified site in the RhCG pore actually recruits NH4(+), which is found in higher concentration and binds with higher affinity than NH3, increasing the efficiency of the transport mechanism. A proton is transferred from NH4(+) to a signature histidine (the only moiety thermodynamically likely to accept a proton) followed by the diffusion of NH3 down the pore. The excess proton is circulated back to the extracellular vestibule through a hydrogen bond network, which involves a highly conserved and functionally important aspartic acid, resulting in the net transport of NH3.

Different hydration patterns in the pores of AmtB and RhCG could determine their transport mechanisms.

The ammonium transporters of the Amt/Rh family facilitate the diffusion of ammonium across cellular membranes. Functional data suggest that Amt proteins, notably found in plants, transport the ammonium ion (NH4(+)), whereas human Rhesus (Rh) proteins transport ammonia (NH3). Comparison between the X-ray structures of the prokaryotic AmtB, assumed to be representative of Amt proteins, and the human RhCG reveals important differences at the level of their pore. Despite these important functional and structural differences between Amt and Rh proteins, studies of the AmtB transporter have led to the suggestion that proteins of both subfamilies work according to the same mechanism and transport ammonia. We performed molecular dynamics simulations of the AmtB and RhCG proteins under different water and ammonia occupancy states of their pore. Free energy calculations suggest that the probability of finding NH3 molecules in the pore of AmtB is negligible in comparison to finding water. The presence of water in the pore of AmtB could support the transport of proton. The pore lumen of RhCG is found to be more hydrophobic due to the presence of a phenylalanine conserved among Rh proteins. Simulations of RhCG also reveal that the signature histidine dyad is occasionally exposed to the extracellular bulk, which is never observed in AmtB. These different hydration patterns are consistent with the idea that Amt and Rh proteins are not functionally equivalent and that permeation takes place according to two distinct mechanisms.

Development of Semiempirical Models for Proton Transfer Reactions in Water

This letter presents a method for the parametrization of semiempirical models for proton transfer reactions in water clusters. Two new models are developed: AM1-W, which is a reparameterization of the classic AM1 model, and AM1PG-W, which is a modified AM1-like model including a pairwise correction to the core repulsion function. Both models show good performance on hydrogen-bonding energies and on proton transfer energy profiles, which are of great importance for proton transfer reactions in large water clusters and in proteins. The parametrization method introduced is general and can be used to develop any other system-specific semiempirical models.

Investigating Ammonium Transport Mechanisms in AmtB and RhCG by Molecular Dynamics Simulations

Membrane proteins of the ubiquitous Amt/Rh family mediate the transport of ammonium. Despite the availability of different X-ray structures that provide many insights on the ammonium permeation process, the molecular details of its mechanism remain controversial. Functional experiments on plant ammonium transporters and rhesus proteins suggest a variety of permeation mechanisms including the passive diffusion of NH3, the antiport of NH4+/H+, the transport of NH4+, or the cotransport of NH3/H+. The X-ray structures have revealed that the pores of the prokaryotic AmtB and the eukaryotic RhCG proteins share a similar architecture suggesting that they might both catalyze the diffusion of NH3. However, molecular mechanics simulations of both proteins reveal that small differences in the pore lining residues might actually alter the properties of the pore. We notably find that the pore of the AmtB transporter can stabilize water molecules at much greater extent than the pore of RhCG. The possible presence of water molecules in the pore lumen of AmtB opens the door to alternative permeation mechanisms, notably involving the co-transport of H+. We discuss the possible permeation mechanisms in both the AmtB and RhCG proteins in light of some recent functional studies, and illustrate how closely related proteins can support quite different mechanisms.

Investigating Co-Transport Mechanisms in the AmtB Ammonium Transporter using QM/MM Molecular Dynamics

AmtB from Escherichia coli is a transmembrane protein with an important role in ammonium transport, especially at low external ammonium concentrations. However, whether AmtB is a channel that permeates NH3 or an NH3/H+ co-transporter is still an open question. An extensive series of hybrid Quantum Mechanical(QM)/Molecular Mechanical(MM) simulations has been performed to investigate the mechanism of ammonium transport through AmtB. Focus has been placed on the deprotonation of ammonium and the possible co-transport of H+ and NH3. Constraint dynamics simulations have been used to obtain the potentials of mean force for the possible NH4+ deprotonation paths involving water molecules and/or protein side chains. Further investigations on the transport pathways of H+ and NH3 have shown the details of the co-transport mechanism. The distribution of solvent and ammonia inside the pore is also analyzed and the possible mechanisms of ammonia re-protonation and how side chains are reset back to original state are presented.