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

• Published in: Biochimica et biophysica acta. Biomembranes 2021-10, Vol.1863 (10), p.183669-183669, Article 183669
• Main Authors: Wege, Stefanie, Qiu, Jiaen, Byrt, Caitlin, Houston, Kelly, Waugh, Robbie, Gilliham, Matthew, Hrmova, Maria
• Format: Article
• Language: English
• Subjects: 3D protein modelling; Confocal microscopy; Molecular dynamics simulations; Salinity; Two-Electrode Voltage-Clamp; α-Helical association and assembly pathways
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2021.183669

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. [Display omitted] •HvHKT1;5 transporters contribute to salinity tolerance of major cereal crops.•M372 deletion in HvHKT1;5 P189 restores plasma membrane localisation but not conductance.•3D models and α-helical assembly pathways of HKT mutants rationalise protein folding.•M372 deletion causes changes in flexibility of α-helices and loops in HvHKT1;5 mutants.