lecture 12

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BioSci M160 / MolBio255 Structure-Function Relationships of Integral Membrane Proteins Lecture 12 Hartmut “Hudel” Luecke Biochemistry, Biophysics & Computer Science Email: hudel@uci.edu http://bass.bio.uci.edu/~hudel






Science (2004) 305: 1587-94. Comment in: Science (2004) 305: 1573-4. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å Khademi S, O'Connell J 3rd, Remis J, Robles-Colmenares Y, Miercke LJ, Stroud RM. Department of Biochemistry and Biophysics, S412C Genentech Hall, University of California-San Francisco, 600 16th Street, San Francisco, CA 94143-2240, USA. The first structure of an ammonia channel from the Amt/MEP/Rh protein superfamily, determined to 1.35 angstrom resolution, shows it to be a channel that spans the membrane 11 times. Two structurally similar halves span the membrane with opposite polarity. Structures with and without ammonia or methyl ammonia show a vestibule that recruits NH4+/NH3, a binding site for NH4+, and a 20 angstrom-long hydrophobic channel that lowers the NH4+ pKa to below 6 and conducts NH3. Favorable interactions for NH3 are seen within the channel and use conserved histidines. Reconstitution of AmtB into vesicles shows that AmtB conducts uncharged NH3. The ammonia transporter


The amino acid sequence of AmtB is arranged topologically as in the structure, with helices viewed as if from inside the channel looking away from the threefold axis. The quasi-twofold axis is perpendicular to the center of the figure. Five helices compose each segment, labeled M1 to M5 and M6 to M10. Related helices M1 and M6, M2 and M7, etc., are boxed in similar colors. Side chains of residues in red circles contribute to the substrate-contacting walls of the channel. Residues in blue circles contribute side chains to the inter-monomer contacts that immediately surround just the threefold axis of the trimer, because many oligomeric membrane proteins either need to insulate against passage of alternate molecules there or use this special location for stability, as in the aquaporins, or for conductance as in K+ channels. The deduced location of the cell membrane is illustrated in gray (35 Å) with light gray for the head group region (to 40 Å thickness).


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


The ammonia transporter Ribbon representation of the AmtB trimer viewed from the extracellular side. Each monomer contains a channel that conducts ammonia. Three NH3 molecules (blue) are inside each channel, while NH4+ ions (orange) remain near the channel entrance. The right monomer has a solvent-accessible transparent surface, colored according to electrostatic potential (red for negative and blue for positive).


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 on top. 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.


Electron density (2Fo-Fc) contoured at 1.5 sigma (blue) for the two-histidine region and surrounding structure, including conserved Asp160 that accepts four short hydrogen bonds (dashed yellow). Additional peaks Am2, Am3, and Am4 seen when crystallized with 25 mM ammonium sulfate are defined in the Fo-Fc omit map at 1.5 sigma (in red), indicating putative NH3 molecule positions (blue spheres). The hydrogen-bonding network shows interactions between His168 and His318 and NH3 peaks in yellow.


Stereo view of the two-histidine center of the channel. Surrounding hydrophobic residues are shown in ball and stick representation. The surface representation covers other surrounding amino acids. Three ammonia-dependent sites are shown (blue spheres) with associated distances (dashed yellow line and yellow labels).


Channel conductance in proteoliposomes. The time course of change in pH inside of vesicles containing CF buffered by 20 mM Hepes as detected by fluorescence change. Initial pH was 6.8 both inside and outside. To initiate flux, 5 mM NH4Cl was added externally to protein-free liposomes (+) and to AmtB-containing proteoliposomes (solid triangles) (protein to lipid ratio was 1: 200 by weight). To control for possible osmotic effects, instead of 5 mM NH4Cl, 5 mM NaCl was added to proteoliposomes (solid circles). The dashed lines are exponential fits to the data.


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.


Crystallographic stats


Perspectives STRUCTURAL BIOLOGY The Atomic Architecture of a Gas Channel Mark A. Knepper & Peter Agre The AmtB ammonia channel of E. coli. The structure of the bacterial integral protein AmtB reveals a wider vestibule at the top and bottom of the channel. The amino acid residues that line the pore of the outer vestibule (Trp148, Phe107, Phe103, and Ser219) stabilize NH4+. Midway through the membrane, the channel narrows over a 20 Å span. Here, two pore-lining residues, His168 and His318, stabilize three NH3 molecules (Am2, Am3, and Am4) through hydrogen bonding (red dashed lines). The molecules return to equilibrium as NH4+ in the inner vestibule.


The structure allows us to deduce how a positively charged ammonium ion is converted to neutral ammonia (a “gas”), makes the transit, and is reconverted to ammonium 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 deduced mechanism has broad implications for “gas channels” in general.


The Rh-related (Rhesus factor) proteins are a family of membrane proteins reported to facilitate the transport of ammonia and carbon dioxide across eukaryotic cell membranes. Human Rh-related proteins are thought to be important in critical physiological processes and, when defective, may result in impairment of systemic pH regulation or central nervous system dysfunction due to ammonium toxicity. The structure of Rh antigens has long been pondered. Now, the trimeric structure of AmtB revealed by Khademi and colleagues suggests a simple explanation for how the three Rh polypeptides of red blood cells (RhAG, RhD, and RhCE) form the Rh antigen complex in the erythrocyte plasma membrane. Rhesus factor

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