A refined atomic scale model of the Saccharomyces cerevisiae K+-translocation protein Trk1p combined with experimental evidence confirms the role of selectivity filter glycines and other key residues

Abstract

Potassium ion (K+) uptake in yeast is mediated mainly by the Trk1/2 proteins that enable cells to survive on external K+ concentration as low as a few μM. Fungal Trks are related to prokaryotic TRK and Ktr and plant HKT K+ transport systems. Overall sequence similarity is very low, thus requiring experimental verification of homology models. Here a refined structural model of the Saccharomyces cerevisiae Trk1 is presented that was obtained by combining homology modeling, molecular dynamics simulation and experimental verification through functional analysis of mutants. Structural models and experimental results showed that glycines within the selectivity filter, conserved among the K-channel/transporter family, are not only important for protein function, but are also required for correct folding/membrane targeting. A conserved aspartic acid in the PA helix (D79) and a lysine in the M2D helix (K1147) were proposed earlier to interact. Our results suggested individual roles of these residues in folding, structural integrity and function. While mutations of D79 completely abolished protein folding, mutations at position 1147 were tolerated to some extent. Intriguingly, a secondary interaction of D79 with R76 could enhance folding/stability of Trk1 and enable a fraction of Trk1[K1147A] to fold. The part of the ion permeation path containing the selectivity filter is shaped similar to that of ion channels. However below the selectivity filter it is obstructed or regulated by a proline containing loop. The presented model could provide the structural basis for addressing the long standing question if Trk1 is a passive or active ion-translocation system.

Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance

“…4) contains an M1 helix, a pore helix (MP), the ion-selectivity filter motif (black asterisks in Fig. 4), and helices M2a and M2b [44]. To facilitate understanding of the identified mutations, we set out to generate a homology model of S. cerevisiae Trk1p ( Sc Trk1p) (Additional file 1: Fig.…”

“…A refined structural model of Trk1 and experimental validations showed that glycines within the selectivity filter are important for protein function and correct folding/membrane targeting [44]. Specifically, mutating these glycines into bulky residues leads to a growth defect in low concentrations of KCl.…”

Monovalent cation transporters at the plasma membrane in yeasts

“…Key residues in that structure have been identified. For example, four Gly within the selectivity filter are thought to be involved in the binding of the cation, and Leu 949 or Lys1147 have been shown to be important in the structure and functioning of the transport process (Haro & Rodriguez‐Navarro, , ; Zayats et al, ). Zayats and coworkers not only published a refined version of the model but also included experimental evidence supporting the structure of Trk1 (Zayats et al, ).…”

“…For example, four Gly within the selectivity filter are thought to be involved in the binding of the cation, and Leu 949 or Lys1147 have been shown to be important in the structure and functioning of the transport process (Haro & Rodriguez‐Navarro, , ; Zayats et al, ). Zayats and coworkers not only published a refined version of the model but also included experimental evidence supporting the structure of Trk1 (Zayats et al, ). A significant difference between ScTrk1 and ScTrk2, or most of the Trk proteins belonging to this family, is the presence of a long intracellular segment (second cytosolic segment) in ScTrk1, whose possible function in regulatory processes has not been definitively established yet.…”

Quantitative description of ion transport via plasma membrane of yeast and small cells

“…Crystal structure of a similar type of transporter, TrkH from Vibrio parahaemolyticus , demonstrates a selectivity filter for K + and Rb + over Na + and Li + (Cao et al, 2011 ). Atomic scale and molecular dynamics modeling of Trk1 from S. cerevisiae confirmed homology with HKT transporters and revealed essential role of glycine residues within potassium selective filter region of the protein, sodium ions were inhibiting for K + transport (Zayats et al, 2015 ). However, still more experimental evidence is required and heterologous expression systems may also be useful for solving the puzzle.…”