Ammonia Channel
Created by Angela Vongphakdy
The ammonia channel (1U7G), AmtB, is a transmembrane protein that is derived from the organism Escherichia Coli and it is involved in the uptake of ammonia across the plasma membrane. The ammonia channel, specifically (1U7G), is a structure of an ammonia channel that has been determined to 1.35-Å resolution from the Amt/MEP/Rh protein family. The molecular weight of the ammonia channel is 39896.41 Da and its isoelectric point is 7.75. Ammonia is one of the most important sources of nitrogen for bacteria, fungi, and plants; but at high levels of concentration, ammonia can be cytotoxic especially for animal cells (6). However, at low levels of concentrations, diffusion becomes limiting for nitrogen uptake and ammonium transport systems are required. The transport of this substance across membranes is therefore of biological relevance because it is vital to the acquirement and metabolism of nitrogen in organisms. Studies of AmtB show that this protein is an ammonia-conducting channel rather than an ammonium ion transporter (3). The most commonly held view concerning ammonia diffusion is that AmtB mediates ammonia uniport activity in an energy-independent, non-concentrative process with movement of NH4+ from the periplasm to the cytoplasm (4,7). Another view is that AmtB could function by bidirectional diffusion of ammonia/ammonium across the membrane (4). The conduction of ammonia/ammonium facilitated by AmtB is concentration dependent. At high extracellular concentrations of ammonium, AmtB transporter activity is inhibited by formation of a complex with a regulatory GlnK protein. At this concentration, passive diffusion across the membrane by NH3 may be sufficient for cell growth. At low concentrations, GlnK does not interact with AmtB and the function of the ammonium channel is activated (4). There are two main views available concerning the nature of the ammonium channel. Some regard AmtB as a secondary transporter that mediates the uptake of the ammonium cation; this view is due to studies that showed the buildup of the substrate analog (methylammonium) within cells in an Amt/MEP-dependent manner. The other, more commonly held view, is that AmtB is merely a channel that increases the rate of equilibration of ammonia across the cytoplasmic membrane (6).
The structure of the ammonia channel can be seen to dictate its function in facilitating the uptake and translocation of ammonia across the membrane. The physiological form of AmtB is a threefold symmetric oligomeric trimer (5). It is a multi-pass and integral transmembrane protein (1). This trimer is made up of 3 identical monomers that consist of 11 transmembrane α-helices, which can be denoted as M1-M11 (in red). The helices are arranged in a right-handed bundle. This protein also contains beta strands (in yellow) and turns (in green) in its secondary structure. Two structurally similar halves span the membrane with opposite polarity where the structures of M1 to M10 show a quasi-twofold axis in the midplane of the membrane that intersects the trimer’s threefold axis. M1-M5 can be seen oriented with opposite polarity to M6-M10. This structural duplication and opposite polarity with respect to the membrane can be found in a number of membrane proteins such as GlpF and aquaporins (5). Indentations in the periplasmic and cytoplasmic surfaces lead into a hydrophobic pore; substrate transport occurs through this narrow, hydrophobic pore located at the center of each monomer of the trimer (6,8).
The ammonia channel (1U7G), observed by Khademi et. al, is an x-ray structure in which ammonia/ammonium molecules are proposed to occupy the ammonia channel. In this model, there is one extraluminal NH4+ molecule at the Am1 position and three intraluminal gaseous NH3 molecules that occupy the Am2, Am3, and Am4 positions respectively. AmtB was crystallized with the mutations Phe-68-Ser, Ser-126-Pro, and Lys-255-Leu. Also, all methionine residues were replaced with selenomethionine (Mse). The crystallization and analysis of this structure resulted in the observation that ammonia/ammonium substrate occupation exists at the binding site and within the lumen of the ammonia channel. This conclusion has been supported and expanded by data that indicate a gradient-driven NH3 uniport movement, either by diffusion of passive transport (5).
There is still debate concerning the specific substrate of the AmtB channel. First, there is controversy over which species of ammonia/ammonium is recognized by AmtB. The prevailing view is that AmtB recognizes and recruits NH4+, but other studies have proposed NH3 recognition as a possibility as well (8). Secondly, there has been additional deliberation about which form of ammonia is conducted within the channel lumen. There is the possibility that NH4+ is recruited and transported undisturbed through out the length of the channel. Another possibility is that NH4+ is deprotonated at some point within the channel so that alone or NH3 in combination with free NH3 is transported as well. The proposed mechanism, made by Khademi et. al, is the ultimate conduction of uncharged NH3 within the hydrophobic lumen of the ammonia pore. This mechanism begins with the recruitment of cationic NH4+ and features its subsequent deprotonation possibly at the periplasmic binding site prior to NH3 conduction within the pore (1). This high-affinity, ion-binding specific site for ammonium is situated at the extracellular pore entrance of the ammonia channel. The degree of high-affinity for ammonia, plays a pivotal role in enhancing net transport at low external ammonium concentrations and acts to discriminate against water. The reconstitution of AmtB into vesicles also confirms that AmtB facilitates the conduction of uncharged NH3 rather than NH4+ (5).
A vestibule recruits NH3/NH4+ at the periplasmic face of the membrane. This vestibule also serves as a binding site for NH4+ and can be denoted as Am1. It has been postulated that the aromatic residues: Phe-103, Phe-107, and Trp-148 define a binding site for NH4+ at the periplasmic pore entrance of the channel (8). The periplasmic side of the channel is sheltered by bothPhe-107 and Phe-215, whose aromatic side chains block direct access to the pore. This obstruction serves to provide selectivity in binding small molecules. The slight barricade in combination with this pore’s high-affinity for ion-binding ensures selectivity for NH4+ and exclusion of water and ions. Deeper into the pore, Asp-160 is found to participate in ordering the surrounding protein structure. Asp-160 also aids in recruiting and binding NH4+ through interactions with NH4+ first by its carbonyl oxygen and then by its carboxylate group (8). The carboxyl group of Asp-160 orients the carbonyl groups of Asp-160, Phe-161, and Ala-162 within the vestibule and makes this periplasmic region cation attracting (5).
Following shortly after this initial extracellular vestibule is the appearance of the first hydrophobic constriction within the channel, Am2, and subsequently after that there is the appearance of a second hydrophobic constriction, Am4. In between the two hydrophobic constrictions lies a 20 Å long hydrophobic channel, Am3. This hydrophobic channel lowers the pka of NH4+ to a value below that of 6 and conducts NH3. The channel features narrow walls and it is mostly nonpolar throughout its length, thus corroborating the conclusion of NH3 conduction. There is a selectivity for NH3 conduction because NH4+ and other ions become increasingly energetically unstable with their approach towards the center of the hydrophobic bilayer whereas NH3 remains relatively stable within this region due to its electrical neutrality (5).
Within the hydrophobic channel are two highly conserved histidine residues, His-318 and His-168 that are located in the middle of the pore. These two residues are linked via a hydrogen bond and they are mutually fixed. The presence of His-318 and His-168 helps foster favorable interactions with NH3 inside the channel and they may also facilitate deprotonation of the ammonium ion when NH4+ enters into the pore. The electrostatic barrier between His-318 and His-168 serve to hinder the permeation of cations, yet still allow the conveyance of NH3 downward into the pore (6). These residues greatly assist the passage of ammonia toward the cytoplasmic end of the channel. The central region of the channel conveys NH3 via an H-bond network created by His-168, His-318, Tyr-32, and the NH3 molecule. In the final stages of transport, Ser-263 plays a pivotal role by acting as a pivoting arm that pushes the NH3 molecule from the cytoplasmic exit gate out into the cytoplasm. NH3 fails to be delivered through the channel if Ser-263 is dysfunctional, which is illustrated by a Ser-263-Ala mutation. Ser-263 is predominantly hydrophilic in comparison to an alanine mutation, which is an aliphatic entity. In this situation, a mutation from serine to alanine at the 263 position hinders the passage of hydrophilic NH3 molecules through the channel toward the cytoplasmic exit region and into the cytoplasm. Ser-263 can aid NH3 in leaving the channel by hydrogen bond formation that serves to essentially drive the movement of NH3 downward to the exit area. The exit gate near the cytoplasmic face is formed by Phe-31, Ile-266, Val-314, and His-318 (4). The cytoplasmic site of the channel is further blocked by Phe-31, which ensures that the channel is kept from becoming hydrated in this region (4).
A number of specific ionic interactions and instances of hydrogen bonding occur within the ammonium channel, some of which have been mentioned earlier. For example, within the initial extracellular vestibule, there are cation-pi interactions with Phe-103, Phe-107, and Trp-148 as well as hydrogen bond interactions with Ser-219. These interactions have been postulated to be a molecular basis of a NH4+ binding site. Also, the two conserved histidine residues, His-318 and His-168, which lie within the hydrophobic pore, are linked by a hydrogen bond. Additionally, the C=O domain of Leu-269 also forms a hydrogen bond with the H of His-318 (5). In the exit gating mechanism, NH3 moves through the channel toward the cytoplasmic pore and exits into the cytoplasm by way of the exit gate, formed by Phe-31, Ile-266, Val-314, and His-318. Hydrogen bonding among these residues, as well as between the two histidine residues, is the most important driving force for ammonium transport through the channel (4).
The ligands within this molecule are NH4+, NH3, and β-octylglucoside. Each ligand serves a different function. The neutral molecule, NH3, moves spontaneously within the channel upon arrival at the Am2 site. The periplasmic entrance rather than the cytoplasmic exit gate determines the function of the ammonium channel as a unidirectional valve for the movement of NH3 to the cytoplasm. There are three intraluminal NH3 molecules positioned at sites within the ammonia channel. NH4+ remains in the extracellular space bound to the first vestibule at the periplasmic face. After NH4+ primary structure homology to AmtB with a PSI-BLAST E-value of 4e-06. PSI-BLAST is a server used to find proteins with similar primary structure to a query protein using a central database. The E-value is assigned based on analysis of total sequence homology. Total sequence homology decreases the E-value while gaps increase the E-value. An E-value less than 0.5 is considered to be significant for proteins. In this case, it is important to keep in mind that the Rh protein was examined to cover 71% of the query and held a max score of 48.4.This protein also shows strong tertiary structure similarities, indicated by a Dali Z score of 40.7. The Dali server retrieves proteins with tertiary similarities to a query protein using a sum-of-pairs method. Structures with significant tertiary similarity and similar folds have a z-score above two. This high z-score is evidenced by an analogous trimeric oligomeric structure found in Rh proteins. Another similarity is the presence of a central pore with the notable occurrence of a twin histidine site in the middle of the pore. A Phe residue also blocks the channel for small molecule transport. Despite several similarities, there are also noteworthy differences. There is a presence of an additional cytoplasmic C-terminal alpha helix as well as a set of residues that may link the C-terminal helix to the Phe blockage, which suggests the possibility of a mechanism where binding of an additional protein to the C terminus could regulate channel opening. There is also the absence of an extracellular pi-cation binding site that is conserved in Amt/MEP structures and the presence of a CO2 binding site near the intracellular exit of the channel has been identified. This binding site contains highly conserved residues that are found in all Rh proteins with the exception of the Rh30 subgroup (9).