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Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori.

Strugatsky D, McNulty R, Munson K, Chen CK, Soltis SM, Sachs G, Luecke H - Nature (2012)

Bottom Line: Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel.Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity.The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

View Article: PubMed Central - PubMed

Affiliation: David Geffen School of Medicine, University of California Los Angeles, Greater West Los Angeles Health Care System, Los Angeles, California 90073, USA.

ABSTRACT
Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

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The Hp UreI urea channel hexamera, Ribbon diagram of hexamer surrounding the lipids of the central bilayer (purple sticks). The C6 hexamer is generated from the three protomers of one asymmetric unit (green) by the crystallographic two-fold axis (second asymmetric unit in teal). b, Electrostatic potential at the periplasmic hexamer surface computed at pH 5.3, the pH at which the crystals were grown (red: -4 kT/e, blue: +4 kT/e). The electrostatic potential was calculated with the program APBSmem22. The green arrow pinpoints the entrance to one of six urea channels.
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Figure 1: The Hp UreI urea channel hexamera, Ribbon diagram of hexamer surrounding the lipids of the central bilayer (purple sticks). The C6 hexamer is generated from the three protomers of one asymmetric unit (green) by the crystallographic two-fold axis (second asymmetric unit in teal). b, Electrostatic potential at the periplasmic hexamer surface computed at pH 5.3, the pH at which the crystals were grown (red: -4 kT/e, blue: +4 kT/e). The electrostatic potential was calculated with the program APBSmem22. The green arrow pinpoints the entrance to one of six urea channels.

Mentions: The structure of HpUreI was determined using multi-wavelength anomalous dispersion (Methods). The structure shows an arrangement of six protomers that form a compact hexameric ring (Fig. 1) about 95 Å in diameter and 45 Å in height. The center of the hexamer is filled with an ordered lipid plug that forms an asymmetric bilayer with electron density for six lipid tails in the periplasmic leaflet and for 18 tails in the cytoplasmic leaflet. This central lipid plug is reminiscent of those reported for other membrane protein oligomers such as bacteriorhodopsin9. The main inter-protomer contacts are between TMH1 and TMH2 of one protomer and TMH3 and TMH4 of a neighboring protomer, a region with conserved residues that are likely important for assembly (Fig. 2a). Native gel electrophoresis of HpUreI confirms that it is a hexamer (Supplementary Fig. 4) in contrast to the previously postulated trimer10. An electron cryomicroscopy 9 Å projection map of a Bacillus cereus amide channel, also a member of the AmiS/UreI family, revealed a similar hexameric arrangement11. Thus, the hexamer likely represents the physiological state rather than a crystallization artifact.


Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori.

Strugatsky D, McNulty R, Munson K, Chen CK, Soltis SM, Sachs G, Luecke H - Nature (2012)

The Hp UreI urea channel hexamera, Ribbon diagram of hexamer surrounding the lipids of the central bilayer (purple sticks). The C6 hexamer is generated from the three protomers of one asymmetric unit (green) by the crystallographic two-fold axis (second asymmetric unit in teal). b, Electrostatic potential at the periplasmic hexamer surface computed at pH 5.3, the pH at which the crystals were grown (red: -4 kT/e, blue: +4 kT/e). The electrostatic potential was calculated with the program APBSmem22. The green arrow pinpoints the entrance to one of six urea channels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3974264&req=5

Figure 1: The Hp UreI urea channel hexamera, Ribbon diagram of hexamer surrounding the lipids of the central bilayer (purple sticks). The C6 hexamer is generated from the three protomers of one asymmetric unit (green) by the crystallographic two-fold axis (second asymmetric unit in teal). b, Electrostatic potential at the periplasmic hexamer surface computed at pH 5.3, the pH at which the crystals were grown (red: -4 kT/e, blue: +4 kT/e). The electrostatic potential was calculated with the program APBSmem22. The green arrow pinpoints the entrance to one of six urea channels.
Mentions: The structure of HpUreI was determined using multi-wavelength anomalous dispersion (Methods). The structure shows an arrangement of six protomers that form a compact hexameric ring (Fig. 1) about 95 Å in diameter and 45 Å in height. The center of the hexamer is filled with an ordered lipid plug that forms an asymmetric bilayer with electron density for six lipid tails in the periplasmic leaflet and for 18 tails in the cytoplasmic leaflet. This central lipid plug is reminiscent of those reported for other membrane protein oligomers such as bacteriorhodopsin9. The main inter-protomer contacts are between TMH1 and TMH2 of one protomer and TMH3 and TMH4 of a neighboring protomer, a region with conserved residues that are likely important for assembly (Fig. 2a). Native gel electrophoresis of HpUreI confirms that it is a hexamer (Supplementary Fig. 4) in contrast to the previously postulated trimer10. An electron cryomicroscopy 9 Å projection map of a Bacillus cereus amide channel, also a member of the AmiS/UreI family, revealed a similar hexameric arrangement11. Thus, the hexamer likely represents the physiological state rather than a crystallization artifact.

Bottom Line: Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel.Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity.The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

View Article: PubMed Central - PubMed

Affiliation: David Geffen School of Medicine, University of California Los Angeles, Greater West Los Angeles Health Care System, Los Angeles, California 90073, USA.

ABSTRACT
Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

Show MeSH
Related in: MedlinePlus