Structure and Function of Transmembrane Segment XII in Osmosensor and Osmoprotectant Transporter ProP of Escherichia coli
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- 作者：Feng Liu ; Doreen E. Culham ; Yaroslava I. Vernikovska ; Robert A. B. Keates ; Joan M. Boggs ; Janet M. Wood
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 15, 2007
- 年：2007
- 卷：46
- 期：19
- 页码：5647 - 5655
- 全文大小：523K
- 年卷期：v.46,no.19(May 15, 2007)
- ISSN：1520-4995
文摘
Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As mediumosmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homologymodel of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein'stopology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain andlinked to TM XII, form an intermolecular, homodimeric 
-helical coiled-coil that tunes the transporter'sresponse to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged,Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with OregonGreen Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except forV429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior ofProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydratedpolar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formedwhen ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TMXII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C renderedProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at highosmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lowerosmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XIIrequired a higher osmolality to activate. They included elements of a GXXXG motif that are predicted toform the interface of TM XII with TM VII. These studies define the position of ProP TM XII within themembrane, further support the predicted structure of ProP, reveal the dimerization interface, and showthat the structure of TM XII influences the osmolality at which ProP activates.
-helical coiled-coil that tunes the transporter'sresponse to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged,Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with OregonGreen Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except forV429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior ofProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydratedpolar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formedwhen ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TMXII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C renderedProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at highosmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lowerosmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XIIrequired a higher osmolality to activate. They included elements of a GXXXG motif that are predicted toform the interface of TM XII with TM VII. These studies define the position of ProP TM XII within themembrane, further support the predicted structure of ProP, reveal the dimerization interface, and showthat the structure of TM XII influences the osmolality at which ProP activates.