Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainly α-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.
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Research Article|
June 27 2012
A urea channel from Bacillus cereus reveals a novel hexameric structure
Gerard H. M. Huysmans;
*Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Nathan Chan;
Nathan Chan
1
†Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
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Jocelyn M. Baldwin;
Jocelyn M. Baldwin
*Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Vincent L. G. Postis;
Vincent L. G. Postis
*Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Svetomir B. Tzokov;
Svetomir B. Tzokov
†Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
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Sarah E. Deacon;
Sarah E. Deacon
‡Institute of Molecular and Cell Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Sylvia Y. M. Yao;
Sylvia Y. M. Yao
§Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7
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James D. Young;
James D. Young
§Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7
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Michael J. McPherson;
Michael J. McPherson
‡Institute of Molecular and Cell Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Per A. Bullough;
Per A. Bullough
3
†Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
3Correspondence may be addressed to either of these authors (email p.bullough@sheffield.ac.uk or s.a.baldwin@leeds.ac.uk).
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Stephen A. Baldwin
Stephen A. Baldwin
3
*Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, U.K.
3Correspondence may be addressed to either of these authors (email p.bullough@sheffield.ac.uk or s.a.baldwin@leeds.ac.uk).
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Publisher: Portland Press Ltd
Received:
January 26 2012
Revision Received:
April 12 2012
Accepted:
May 03 2012
Accepted Manuscript online:
May 03 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 445 (2): 157–166.
Article history
Received:
January 26 2012
Revision Received:
April 12 2012
Accepted:
May 03 2012
Accepted Manuscript online:
May 03 2012
Citation
Gerard H. M. Huysmans, Nathan Chan, Jocelyn M. Baldwin, Vincent L. G. Postis, Svetomir B. Tzokov, Sarah E. Deacon, Sylvia Y. M. Yao, James D. Young, Michael J. McPherson, Per A. Bullough, Stephen A. Baldwin; A urea channel from Bacillus cereus reveals a novel hexameric structure. Biochem J 15 July 2012; 445 (2): 157–166. doi: https://doi.org/10.1042/BJ20120169
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