Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17
Year: 2016
Type: article
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. • Phyre2 and I-TASSER predicted the 3D structure of a plant transporter AtCHX17 as similar to bacterial Na + /H + antiporters. • The distinct protein architecture highlighted non-conserved and conserved residues potentially critical for activity. • Mutagenesis of residues in the center and at unwound regions of two transmembrane helices resulted in loss of CHX17 activity. • Results identify D201, N200, T170 and K383 as core residues in translocation, similar to characterized cation/H + exchangers. Acidic residue(s) near the transporter surface may sense pH and regulate activity.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,870
OpenAlex ID: W2354460888