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. 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.
authors.affiliation: Department of Cell Biology and Molecular Genetics (DC, SP, ZR, HS), University of Maryland, College Park, MD 20742, USA.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Czerny
authors.affiliation: Department of Cell Biology and Molecular Genetics (DC, SP, ZR, HS), University of Maryland, College Park, MD 20742, USA.
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Padmanaban
authors.affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA.
authors.forename: Andriy
authors.initials: A
authors.lastname: Anishkin
authors.affiliation: Dpto de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, c/. Profesor Albareda 1, 18008Granada, Spain.
authors.forename: Kees
authors.initials: K
authors.lastname: Venema
authors.affiliation: Department of Cell Biology and Molecular Genetics (DC, SP, ZR, HS), University of Maryland, College Park, MD 20742, USA.
authors.forename: Zoya
authors.initials: Z
authors.lastname: Riaz
authors.affiliation: Department of Cell Biology and Molecular Genetics (DC, SP, ZR, HS), University of Maryland, College Park, MD 20742, USA; Maryland Agricultural Experiment Station, Department of Plant Science and Landscape Architecture (HS), University of Maryland, College Park, MD 20742, USA. Electronic address: hsze@umd.edu.
authors.forename: Heven
authors.initials: H
authors.lastname: Sze
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; C493886:CHX17 protein, Arabidopsis; D017923:Sodium-Hydrogen Exchangers
keywords: Arabidopsis; Cation/proton antiporter; Core residues; Structure modeling; Transporter; Yeast expression; pH regulation