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Title: Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17

Abstract

Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na+/H+ antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. Here, these results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates inmore » ion recognition, coordination and/or translocation, similar to characterized cation/H+ exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Maryland, College Park, MD (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Contributing Org.:
University of Maryland
OSTI Identifier:
1325359
Alternate Identifier(s):
OSTI ID: 1351089
Grant/Contract Number:  
BES DEFG-0207ER15883; BIO2012-33655; BIO2015-65056; FG02-07ER15883
Resource Type:
Published Article
Journal Name:
Biochimica et Biophysica Acta. Biomembranes
Additional Journal Information:
Journal Name: Biochimica et Biophysica Acta. Biomembranes Journal Volume: 1858 Journal Issue: 9; Journal ID: ISSN 0005-2736
Publisher:
Elsevier
Country of Publication:
Netherlands
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; cation/proton antiporter; potassium; structure modeling; transporter; pH regulation; yeast expression; Arabidopsis; core residues

Citation Formats

Czerny, Daniel D., Padmanaban, Senthilkumar, Anishkin, Andriy, Venema, Kees, Riaz, Zoya, and Sze, Heven. Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17. Netherlands: N. p., 2016. Web. https://doi.org/10.1016/j.bbamem.2016.05.008.
Czerny, Daniel D., Padmanaban, Senthilkumar, Anishkin, Andriy, Venema, Kees, Riaz, Zoya, & Sze, Heven. Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17. Netherlands. https://doi.org/10.1016/j.bbamem.2016.05.008
Czerny, Daniel D., Padmanaban, Senthilkumar, Anishkin, Andriy, Venema, Kees, Riaz, Zoya, and Sze, Heven. Thu . "Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17". Netherlands. https://doi.org/10.1016/j.bbamem.2016.05.008.
@article{osti_1325359,
title = {Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17},
author = {Czerny, Daniel D. and Padmanaban, Senthilkumar and Anishkin, Andriy and Venema, Kees and Riaz, Zoya and Sze, Heven},
abstractNote = {Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na+/H+ antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. Here, these results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H+ exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.},
doi = {10.1016/j.bbamem.2016.05.008},
journal = {Biochimica et Biophysica Acta. Biomembranes},
number = 9,
volume = 1858,
place = {Netherlands},
year = {2016},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
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https://doi.org/10.1016/j.bbamem.2016.05.008

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