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 permeases͞ammonium transporters͞rhesus) 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-Å 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 permeases͞ammonium transporters͞rhesus 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.conformational change ͉ x-ray structure

Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells

“…The conclusion that RhBG and RhCG carry out NH 3 transport fully correlates with our recent study demonstrating that the rate constant of NH 3 movement in RBCs is strictly dependent on RhAG expression level [20]. These results, supported by crystallographic and functional studies demonstrating that AmtB, a bacterial member of the Amt/Mep/Rh superfamily, is a channel that conducts NH 3 [30,31], first identified Rh glycoproteins as members of an NH 3 channel family in mammals. In support of a physiological relevance for the capacity of RhBG and RhCG to transport NH 3 , it has been shown, at least in the kidney, that epithelial cells that exhibit the highest NH 3 permeability, namely intercalated cells of the collecting tubules [32], are those which express RhBG and RhCG [9][10][11].…”

“…However, based on the relatively restricted organ and tissue distribution of Rh proteins and the observation that mutants of Chlamydomonas reinhardtii lacking the paralogue Rh1 protein grow very slowly under high CO 2 conditions, these authors further suggested that mammalian Rh proteins might function as CO 2 rather than as NH 3 gas channels [42,43]. Although the narrowness of the channel in AmtB and the nature of the porelining amino acids [30,31] do not favour the hypothesis of a CO 2 transporter, we cannot formally rule out that Rh glycoproteins are gas channels that might facilitate either NH 3 or CO 2 transport depending on which cell types they are expressed in. While there is increasing evidence for the existence of conserved NH 3 gas channels in bacteria to mammals, the integrated biological role of Rh glycoproteins in mammal physiology remains unknown.…”

“…It is tempting to speculate that structural differences within the vestibule located at the top of the narrow channel of RhBG and RhCG, as hypothesized from the crystal structure of AmtB [30,31], should account for these different substrate accessibilities.…”

Rhesus glycoprotein gene expression in the mangrove killifishKryptolebias marmoratusexposed to elevated environmental ammonia levels and air

“…RhAG expression is restricted to the erythrocyte membrane, whereas RhBG and RhCG are expressed in various tissues in the mammalian systems, including liver, kidney and gastrointestinal tract (Handlogten et al, 2005;Liu et al, 2000;Liu et al, 2001;. Studies in mammalian and plant systems reveal that at least some of those Rh genes encode proteins that mediate NH 3 /NH 4 + movement (Khademi et al, 2004;Zheng et al, 2004;Mayer et al, 2006), but the form of ammonia (NH 3 gas or NH 4 + ion) being transported, and whether the transport is active or passive, are still under much debate (Bakouh et al, 2004;Khademi et al, 2004;Nakhoul et al, 2005). A fulllength cDNA of Rh-like protein (Rh-CM) has been identified recently from the gills of the aquatic crab Carcinus maenas, which has a similar predicted transmembrane structure as the mammalian Rh proteins (Weihrauch et al, 2004).…”

Protéines de la famille Rh et transport membranaire du gaz NH3

“…La protéine RhAG serait donc l'une des premières protéines membranaires fonctionnant comme un canal à gaz, dont la conductance approximative (gradient 1 M d'ammonium, pH 7) a été estimée à 2x10 6 molécules NH 3 Une contribution essentielle a été apportée par d'autres travaux très récents qui ont révélé la structure cristallographique 3D à haute résolution du transporteur d'ammonium AmtB de E. coli [4,5]. Ces études ont montré que chaque sous-unité du complexe trimé-rique AmtB présente la même conformation en présence et en l'absence de substrat (ammonium et méthylammonium), suggérant que cette protéine fonctionne comme un canal plutôt que comme un transporteur, car un mécanisme de transport implique classiquement l'existence de différents états de conformation induits par la translocation du substrat.…”