Simulation of pH-Dependent Conformational Transitions in Membrane Proteins: The CLC-ec1 Cl-/H+ Antiporter.

Ekaterina Kots,Harel Weinstein,Derek M Shore

doi:10.3390/molecules26226956

Abstract

Intracellular transport of chloride by members of the CLC transporter family involves a coupled exchange between a Cl− anion and a proton (H+), which makes the transport function dependent on ambient pH. Transport activity peaks at pH 4.5 and stalls at neutral pH. However, a structure of the WT protein at acidic pH is not available, making it difficult to assess the global conformational rearrangements that support a pH-dependent gating mechanism. To enable modeling of the CLC-ec1 dimer at acidic pH, we have applied molecular dynamics simulations (MD) featuring a new force field modification scheme—termed an Equilibrium constant pH approach (ECpH). The ECpH method utilizes linear interpolation between the force field parameters of protonated and deprotonated states of titratable residues to achieve a representation of pH-dependence in a narrow range of physiological pH values. Simulations of the CLC-ec1 dimer at neutral and acidic pH comparing ECpH-MD to canonical MD, in which the pH-dependent protonation is represented by a binary scheme, substantiates the better agreement of the conformational changes and the final model with experimental data from NMR, cross-link and AFM studies, and reveals structural elements that support the gate-opening at pH 4.5, including the key glutamates Gluin and Gluex.

Highlights

Members of the CLC family of transmembrane proteins that mediate voltage-dependent transport of chloride across the membrane cell offer powerful examples of the key role that the ambient pH can play in the mechanisms of function of biomolecular systems in the cell membranes
The structural rearrangements underlying the pH-dependent transition of the CLC-ec1 dimer between opened and closed gate states observed with High-Resolution AFM (HR-AFM) [19] and a variety of other experimental approaches [18,21,22,23,24] were investigated with molecular dynamics simulations (MD) simulations employing the Equilibrium constant pH approach (ECpH) force field modification scheme. This approach enables ECpH to overcome the binary representation of the protonation states in the force field of cMD, thereby introducing a crucial improvement in the ability of MD simulations to represent protein molecules in a range of physiological pH (Appendix A)
Application of ECpH to model pH-dependent conformational changes of the CLC-ec1 transporter produced a detailed representation of the complex structural response to the change from neutral to acidic pH

Summary

Introduction

Members of the CLC family of transmembrane proteins that mediate voltage-dependent transport of chloride across the membrane cell offer powerful examples of the key role that the ambient pH can play in the mechanisms of function of biomolecular systems in the cell membranes. The CLCs family comprises both ion channels and transporters that share major structural motifs [1,2]. Nine different human CLCs were identified, expressed in brain, heart, liver, gut and muscle tissues [3,4]. They have major physiological roles in maintaining homeostasis (e.g., in skeleton muscle tissues, the CLC-1 channel supports action potential generation by sodium and potassium re-polarizing the muscle fiber) [5,6]. The prokaryotic analog of human CLC transporters, CLC-ec, has been studied extensively as a mechanistic and structural prototype for the human transporters in this class [7,8,9,10]

Methods

Results

Conclusion