|How the Membrane Protein AmtB Transports Ammonia|
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.”
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.
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.
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).