The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli

Abstract

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however, functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH(4)(+) or NH(3)) of the transported species. We have solved the structure of wild-type AmtB from Escherichia coli in two crystal forms at 1.8- and 2.1-A resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB. At the periplasmic entry, a binding site for NH(4)(+) is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent, with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH(3.) The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transporters/rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner.

Fig. 1.

Overall structure of the AmtB trimer. (a) Surface view of the AmtB trimer from the extracellular side. Two monomers of an AmtB trimer are drawn in salmon and slate; one monomer is shown as a ribbon. (b) Side view of the molecular surface of the AmtB trimer.

Fig. 2.

Topological representation of the AmtB monomer. (a) Side view from the membrane. (b) Top view from the extracellular side.

Fig. 3.

Sequence alignment of selected members of the Mep/Amt/Rh family. Secondary structure according to dspp (28, 52) and sequence numbering (AmtB of E. coli) are indicated. Highly conserved residues are in red. Amino acid sequences are as follows: AmtB, E. coli (P37905); AmtA, Dictyostelium discoideum (BAB39709); Amt, Schizosaccharomyces pombe (NP_593983); Amt, Chlamydomonas reinhardtii (AAL38652); AtAMT1, Arabidopsis thaliana (P54144); and RhAG, Homo sapiens (Q02094). The alignment was made with multalin (53) and adjusted manually based on the structural information

Fig. 4.

Ammonium-binding site and hydrophobic pore. (a) Stereoview from the extracellular side of the putative substrate-binding site at the extracellular pore entry. The 2 F_o–F_c electron density map of AmtB is contoured at 1.0 σ in slate; the F_o–F_c electron density map is contoured at 3.0 σ in yellow. (b) A cut through the AmtB monomer surface within the plane of the membrane. The dark elongated feature in the center indicates the location and shape of the conduction pore. The pore is seen to be blocked by F215 and F104 on its extracellular side, and H168 and H318 are seen to be adjacent in its central part. (c) Stereoview of the AmtB pore-lining residues in the R3 structure. Highly conserved residues (Fig. 3) are shown in salmon, less conserved ones in green. A final omit difference electron density (F_o–F_c) contoured at 3.0 σ is shown in the pore region in red. (d) Representation as in c for the P6₃ structure (crystal grown in the absence of ammonium sulfate). Note the different structure at the cytoplasmic exit at V314 and the different peaks in the omit difference density.

Fig. 5.

Conformational change at the cytoplasmic exit between R3 and P6₃ crystal structures. The pore is viewed from the cytoplasmic side. (a) R3 structure (“open” exit conformation). (b)P6₃ structure (“closed” exit conformation).