A single residue deletion in the barley HKT1;5 P189 variant restores plasma membrane localisation but not Na+ conductance

Stefanie Wege, Jiaen Qiu, Caitlin Byrt, Kelly Houston, Robbie Waugh, Matthew Gilliham, Maria Hrmova (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

Abstract

Leaf Na+ exclusion, mediated by plasma membrane-localised Class 1 High-affinity potassium (K+) Transporters (HKTs), is a key mechanism contributing to salinity tolerance of several major crop plants. We determined previously that the leucine to proline residue substitution at position 189 (L189P) in barley HvHKT1;5 disrupts its characteristic plasma membrane localisation and Na+ conductance. Here, we focus on a surprising observation that a single residue deletion of methionine at position 372 (M372del) within the conserved VMMYL motif in plant HKTs, restores plasma membrane localisation but not Na+ conductance in HvHKT1;5 P189. To clarify why the singular M372 deletion regains plasma membrane localisation, we built 3D models and defined α-helical assembly pathways of the P189 M372del mutant, and compared these findings to the wild-type protein, and the HvHKT1;5 L189 variant and its M372del mutant. We find that α-helical association and assembly pathways in HvHKT1;5 proteins fall in two contrasting categories. Inspections of structural flexibility through molecular dynamics simulations revealed that the conformational states of HvHKT1;5 P189 diverge from those of the L189 variant and M372del mutants. We propose that M372del in HvHKT1;5 P189 instigates structural rearrangements allowing routing to the plasma membrane, while the restoration of conductance would require further interventions. We integrate the microscopy, electrophysiology, and biocomputational data and discuss how a profound structural change in HvHKT1;5 P189 M372del impacts its α-helical protein association pathway and flexibility, and how these features underlie a delicate balance leading to restoring plasma membrane localisation but not Na+ conductance.

Original languageEnglish
Article number183669
Number of pages11
JournalBBA - Biomembranes
Volume1863
Issue number10
Early online date15 Jun 2021
DOIs
Publication statusE-pub ahead of print - 15 Jun 2021

Keywords

  • 3D protein modelling
  • Confocal microscopy
  • Molecular dynamics simulations
  • Salinity
  • Two-Electrode Voltage-Clamp
  • α-Helical association and assembly pathways

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