Genetic Evidence for an Essential Oscillation of Transmembrane-Spanning Segment 5 in the Escherichia coli Ammonium Channel AmtB
| Autor: | Jason A. Hall, Kwang-Seo Kim, William Inwood, Sydney Kustu, Rebecca Fong |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Protein Conformation PII Nitrogen Regulatory Proteins Molecular Sequence Data Mutant Investigations Biology Suppression Genetic Protein structure ATP-Dependent Proteases Escherichia coli Genetics Amino Acid Sequence Cation Transport Proteins Peptide sequence Sequence Deletion Escherichia coli Proteins C-terminus Cell Membrane Periplasmic space Nucleotidyltransferases Transmembrane protein Transport protein Cytoplasm Isotope Labeling Electrophoresis Polyacrylamide Gel |
| Zdroj: | Genetics. 183:1341-1355 |
| ISSN: | 1943-2631 |
| DOI: | 10.1534/genetics.109.109579 |
| Popis: | Ammonium channels, called Amt or Mep, concentrate \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{NH}}_{4}^{+}\end{equation*}\end{document} against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear. We have described lesions in conserved C-terminal residues that inactivate AmtB and here characterize 38 intragenic suppressors upstream of the C terminus (∼1/3 of total suppressors). Three that occurred repeatedly, including the previously characterized W148L at the pore entry, restored growth at low NH3 to nearly wild-type levels and hence restored high activity. V116L completely restored function to two of the mutant proteins and, when separated from other lesions, did not damage wild-type AmtB. A179E notably altered folding of AmtB, compensated for all inactivating C-terminal lesions, and damaged wild-type AmtB. V116L and A179E lie at the cytoplasmic end of transmembrane-spanning segments (TM) 3 and 5, respectively, and the proximal part of the C-terminal tail makes intimate contacts with the loops following them before crossing to the adjacent monomer. Collectively, the properties of intragenic suppressor strains lead us to postulate that the C-terminal tail facilitates an oscillation of TM 5 that is required for coordinated pore function and high AmtB activity. Movement of TM 5 appears to control the opening of both the periplasmic entry and the cytoplasmic exit to the pore. |
| Databáze: | OpenAIRE |
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