Ammonia channel AmtB (PDB ID:
1U7G) from Escherichia coli
Created by: Michael Lingelbach
Ammonia channel AmtB (PDB ID: 1U7G, AmtB) from Escherichia coli is a transmembrane channel protein that facilitates ammonia transport, a fundamental component of nitrogen metabolism for all domains of life. In bacteria and fungi under normal conditions, the passive membrane penetration of ammonia provides a sufficient source of inorganic nitrogen; AmtB is necessary in ammonia-poor conditions when the rate of diffusion is insufficient for nitrogen metabolism. In animals, high levels of ammonia are cytotoxic (1,2). Animals possess proteins functionally related to AmtB that eliminate excess ammonia from the cytoplasm; human rhesus glycoprotein RhCG (PDB ID: 3HD6, RhCG) from Homo sapiens is one example of an animal ammonia channel (3). AmtB was the first structurally studied transmembrane channel protein capable of conducting unhydrated molecules that are gaseous in isolation. Therefore, information gained through the study of AmtB is useful in the study of the closely related Rh family (1, 2, 4).
AmtB crystallized as hexagonal crystals via hanging drop vapor diffusion at room temperature;
the well buffer contained polyethylene glycol and 0.1 M 2-(N-morpholino) ethanesulfonic
acid at a pH of 6.5. The ligands ammonia, ammonium, and
n-octyl-β•D-glucopyranoside (OG) appear in the crystal structure. Ammonia and
ammonium are the substrates of AmtB; the channel transports these substrates across
the cellular membrane. The presence of OG is an artifact of the protein
purification process; the protein was first solubilized in 200 mM OG before
purification using Ni-affinity chromatography, and the collected solution was filtered
using gel filtration with 40 mM OG. The crystal structure was determined to a
resolution of 1.35 Å using single wavelength x-ray diffraction. The AmtB crystallized is
not the wild type; the protein contains the mutations S68F, P126S, and L255K (1).
Physiologically, AmtB exists as a homotrimer consisting
of 385 residues per subunit (Figure 1). Each subunit of AmtB contains two beta sheets and 11 transmembrane-spanning α-helices, M1 to M11 (Figure 2). The three subunits associate through hydrophobic interactions
between residues from helices M1, M6, M7, M8, and M9 from one monomer with the
residues from helices M1, M2, and M3 in the adjacent monomer to form the
homotrimer. The homotrimer has a diameter of 81 Å in the transmembrane region
and a depth of approximately 65 Å (1). Each subunit has a molecular weight of 39,896.41 Da and an isoelectric
point of 7.75 according to the ExPASy molecular
weight and isoelectric point tool
(5).
On
the extracellular side of the homotrimer, the three M1 helices cluster to block
the conduction of molecules along the central axis. AmtB only conducts
molecules through the pores of the subunits (4). On the cytoplasmic side of the homotrimer, the M1
and M6 helices of the three subunits form a pocket 10Å in diameter. Tyr-62 at the periplasmic side and Tyr-180, Trp-250, and Trp-297
on the cytoplasmic side form the membrane-aqueous interface. The helices M1 to
M10 diverge from a central axis in a right-handed helical bundle; the bundle
creates one vestibule on the periplasmic and one vestibule on the cytoplasmic
surface (1, 2, 4). The helices form a
pore between the periplasmic and cytoplasmic vestibules that conducts ammonia (1).
Asp-160 is critical to the structure of the pore; when Asp-160 mutates to Ala-160, AmtB
no longer transports methylammonium. Asp-160 accepts hydrogen bonds at the Oδ2
from the N-H of Thr-165, the OγH of Thr-165, the N-H of Gly-164, and the N-H of
Gly-163. The Asp-160 carboxyl group aligns the helices M2, M4, and M6, the
location of the carbonyl groups of Asp-160, Phe-161, Ala-162, Ser-68, Ser-219,
Val-147, and Trp-148. These carbonyl groups create a net negative dipole at the
periplasmic vestibule that attracts cations (1, 2, 4).
The periplasmic ammonium-binding
site is located at the base of the periplasmic vestibule; ammonium interacts
via hydrogen bonding with the hydroxyl group of Ser-219 and via π -cation interactions with the aromatic rings of Phe-107 and Trp-148 (1, 4). These interactions stabilize ammonium via
tetrahedral coordination. Deeper in the vestibule, the
π –face of Phe-215 restricts access to the channel from the periplasmic vestibule of each subunit
(Figure 3). Phe-215 acts to deprotonate ammonium to form neutral ammonia for
transport; a mutation in Phe-215 results in an open yet inactive channel (1, 4).
The ammonia channel contains two
constricted hydrophobic regions that select for ammonia over ammonium and other
charged molecules. Unprotonated His-168 Nδ1 and His-318 Nδ1H, bound via hydrogen
bond, create the first hydrophobic region that extends approximately 20 Å (Figure
4). Throughout this constriction the channel walls are predominantly nonpolar
and narrow. The nonpolar span lowers the acid dissociation constant of ammonium,
encouraging the formation of ammonia at physiological pH (2). Interactions with the side chains of His-168 and
His-318 stabilize ammonia; the electrostatic barrier formed by His-168 and
His-318 inhibits the transmission of cations and selects for neutral molecules (1).
Trp-148, Phe-103, Phe-161, and Tyr-140 form the second hydrophobic constriction in the channel via π-cation
interactions with ammonium; this interaction again increases selectivity for
ammonia transport. The transport of ammonia does not require replacement of a
hydration shell; therefore the channel avoids the energetic cost of replacing a
water of hydration that ammonium transport would incur. The two constricted
hydrophobic regions in the channel are crucial to preventing the transport of
charged cations across the cell membrane. Although potassium channels conduct ammonium,
Amt/MEP proteins are not capable of transporting potassium. Therefore, ammonia
channels of the Amt/MEP/Rh variety do not disrupt the balance of membrane
potential in eukaryotes (1, 2).
The signal transduction protein GlnK (PDB ID: 1GNK) from Escherichia coli regulates the activity of AmtB by rapidly binding to AmtB reversibly in the
presence of high extracellular levels of ammonium (Figure 5). GlnK
exists physiologically as homotrimer with each subunit possessing three loops
designated as B, C, and T. In the GlnK-AmtB complex (PDB ID: 2NUU), GlnK
interacts exclusively with AmtB via Tyr-51, a highly conserved residue found in
the T loop (Figure
6). The
two proteins bind with a stoichiometric ratio of 1:1; the binding of GlnK and
AmtB alters the conformation of AmtB on the cytoplasmic face, preventing
transmission of ammonia (6, 7).
Human rhesus glycoprotein RhCG from Homo sapiens is, like AmtB, a
homotrimeric ammonia transport protein. Both proteins belong to the Amt/MEP/Rh family; the proteins are structurally and
functionally similar. The Dali server uses a sum-of-pair method to assign a
Z-score by comparing intramolecular distances; tertiary structures with a
Z-score of 2 or greater show significant similarity. The tertiary structures of
AmtB and RhCG have a Z-score of 39.7. AmtB
has alpha helices up to 5 residues longer than RhCG. The observed coils found
in AmtB contain on average more residues than counterparts in RhCG. AmtB contains
more beta strands than RhCG (8).
RhCG contains one additional transmembrane
alpha helix in comparison to AmtB. The n-terminal region that forms the 12th
helix in RhCG is not present in AmtB because the region in AmtB is cleaved
after acting as a signal sequence (1, 3).
PSI-BLAST compares the sequence of a
user provided query to the online protein database in order to find proteins with
similar primary structures. PSI-BLAST assigns an E value, derived from the
presence of gaps, as an indicator of sequence similarity. Gaps consist of one
or multiple amino acids present in a subject yet not present in a query sequence
with homology continuing after the gap. RhCG
and AmtB have an E value of 1e-05; an E value of 0.05 or less is a significant
indicator of sequence similarity (9).
RhCG and AmtB both function as
ammonia transporters. In the periplasmic vesicle, RhCG possesses aliphatic
residues whereas AmtB contains aromatic residues. This difference reflects the difference in substrates that AmtB and RhCG transport; AmtB recruits and deprotonates ammonium for
transport whereas RhCG transports neutral ammonia. The environment of E. coli lacks high concentrations of
ammonium; therefore, the periplasmic binding site of AmtB actively recruits
ammonium to sustain nitrogen metabolism. H.
sapiens do not rely on the nitrogen metabolism and must eliminate
nitrogenous waste; RhCG facilitates ammonia homeostasis and acid-base
regulation (10, 11).