LBNL Masthead A-Z IndexBerkeley Lab mastheadU.S. Department of Energy logoPhone BookJobsSearch
How the Membrane Protein AmtB Transports Ammonia Print

Membrane proteins provide molecular-sized entry and exit portals for the various substances that pass into and out of cells. While life scientists have solved the structures of protein channels for ions, uncharged solutes, and even water, up to now they have only been able to guess at the precise mechanisms by which gases (such as NH3, CO2, O2, NO, N2O, etc.) cross biological membranes. But, with the first high-resolution structure of a bacterial ammonia transporter (AmtB), determined by a team in the Stroud group from the University of California, San Francisco, it is now known that this family of transporters conducts ammonia by stripping off the proton from the ammonium (NH4+) cation and conducting the uncharged NH3 “gas.”

A Doorway for Letting Ammonia into Cells

Like any factory, a biological cell takes in raw materials and energy and expels waste products. What goes in and out passes through the cell membrane that surrounds the cell’s interior via channels and transporters that serve as carefully guarded entry and exit ports. Determining the molecular-scale mechanisms by which the membrane proteins accomplish these roles is a major challenge among life scientists. Achieving this goal relies on atomic structures of such membrane proteins obtained from x-ray crystallography. The difficulty of growing robust crystals of membrane proteins for crystallography has limited progress, so that relatively few structures are known relative to those of soluble proteins.

After successfully growing the necessary crystals, Khademi et al. determined the first atomic structure of a channel (known as AmtB) that passes ammonia gas molecules through the bacterial cell membrane of E. coli. The structure allowed them to deduce how a positively charged ammonium ion is converted to neutral ammonia (a “gas”), makes the transit, and is reconverted without requiring an energy source to drive the process and without altering the electrical potential (voltage) that exists across the cellular membrane. Since this membrane protein is related to similar ones in higher organisms, including the Rh factors in humans, the mechanism they deduced has broad implications for “gas channels” in general.

Ribbon representation of the AmtB trimer is viewed from the extracellular side. Each monomer contains a channel that conducts ammonia. Three NH3 molecules (blue) are in each channel, while NH4+ ions (orange) remain near the channel entrance.

Progress in determining structures of membrane proteins of all kinds has been slowed by the difficulty of obtaining sufficiently robust crystals that diffract to high resolution. A common strategy is to grow crystals of proteins from multiple organisms in which the protein is known to have evolved from a common ancestor (orthologs) and select the one that gives the best diffraction data. The UCSF researchers cloned multiple orthologs of the integral membrane protein AmtB belonging to the Amt/MEP/Rh superfamily.

To define any preferred sites for ammonia or methyl ammonia (CH3NH2) and to clarify the mechanism for transport or conductance of these molecules, crystals were grown in the absence of any ammonium derivative and in the presence of ammonium sulfate or methyl ammonium sulfate.

A stereo view of the monomeric ammonia channel viewed down the quasi-twofold axis. Corresponding related helices are shown in the same color. The extracellular side is uppermost. The brown rectangle represents the inferred position of the hydrophobic portion of the bilayer. Three NH3 molecules seen only when crystallized in presence of ammonium sulfate are shown as blue spheres. The orange sphere represents an NH4+ ion at the vestibule.

Diffraction data from crystals of AmtB from the bacterium Escherichia coli were collected at ALS Beamline 8.3.1 with a CCD area detector. Phases were calculated from multiple-wavelength anomalous dispersion (MAD) data from a selenomethionine (SeMet)-substituted protein. After data processing (solvent flattening and phase extension to 2.0 Å), the model was refined to 1.35 Å, the highest-resolution structure of any membrane protein to date.

Overall, the structure shows that AmtB is a trimer, with each monomer containing a channel conducting ammonia. The monomer protein chain includes two structurally similar motifs of opposite polarity. Each motif spans the cell membrane between the periplasm (region between the cell wall and the membrane) and the cytoplasm (cell interior) five times.

Comparison of the structures with and without ammonia and with methyl ammonia enabled the team to identify a wider vestibule site at the periplasmic side of the membrane that recruits NH4+ and a narrower 20-Å-long hydrophobic channel midway through the membrane that lowers the dissociation constant of NH4+, thereby forming NH3, which is then stabilized by interactions with two conserved histidine side chains inside the channel. In a second vestibule at the cytoplasmic end of the channel, the NH3 returns to equilibrium as NH4+. An ammonia conduction assay was devised using stopped-flow kinetics and, together with the structural result, proved that it is only neutral NH3 that is conducted by the channel. This is the first time that the structure and mechanism of a “gas channel” has been determined.

Conductance of uncharged NH3, versus the NH4+ ion, solves several biological problems. Transport of only uncharged NH3 assures selectivity against all ions. NH4+ or any other ion would be unstable in the center of the hydrophobic bilayer, while NH3 is not. Passage of uncharged NH3 would not result in a net change of protons across the membrane nor would it change the membrane potential, thus neither energy any negative counter ion to balance the charge is needed to accumulate ammonia.

Summary of mechanism of conductance. Two vestibules reside at the top and bottom of the channel. Amino acid residues (blue, red, and gray ball-and-stick models) that line the pore of the outer vestibule stabilize NH4+ (green and yellow). After a proton (orange) departs, the channel narrows midway through the membrane for a 20-Å distance and is hydrophobic. Here, two pore-lining histidine residues (light and dark blue) stabilize three NH3 molecules through hydrogen bonding. Farther on, with the addition of a proton (orange), the molecules return to equilibrium as NH4+ in the inner vestibule.

The structure of AmtB and the mechanism of gas transport are common to other members of the superfamily in eukaryotic cells. For example, related Rh proteins in humans are thought to be critical players in systemic pH regulation in the kidney, in amino acid biosynthesis, and in the central nervous system.

Research conducted by S. Khademi, J. O’Connell III, J. Remis, Y. Robles-Colmenares, L.J.W. Miercke, and R.M. Stroud (University of California, San Francisco).

Research funding: National Institutes of General Medical Sciences. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: S. Khademi, J. O'Connell III, J. Remis, Y. Robles-Colmenares, L.J. Miercke, and R.M. Stroud, “Mechanism of ammonia transport by Amt/MEP/Rh: Structure of AmtB at 1.35 Å,” Science 305, 1587 (2004).