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

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.

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