Unexpected Gating Behaviour of an Engineered Potassium Channel Kir.

Charline Fagnen,Ludovic Bannwarth,Iman Oubella,Dania Zuniga,Eric Forest,Rosa Scala,David Perahia,Samuel Guilbault,Rita De Zorzi,Catherine Vénien-Bryan,Saïd Bendahhou

doi:10.3389/fmolb.2021.691901

Abstract

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.

Highlights

A detailed study of function requires careful dissection of the mechanistic steps
The theoretical results presented are based on Molecular Dynamics using excited Normal Modes (MDeNM) (Molecular Dynamics with excited Normal Modes) simulations in which different linear combinations of a selected set of normal modes (NMs) related to the opening/ closing of the channel are excited in molecular dynamics (MD) simulations (Costa et al, 2015)
Through such a combined use of both methods, MDeNM allows a realistic exploration of the normal mode space relevant for the opening/closing mechanism taking into account the full environment of the protein at the ambient temperature

Summary

Introduction

A detailed study of function requires careful dissection of the mechanistic steps. Protein engineering can provide a powerful tool for studying the relationships between structure and function. This structure allowed proposing a mechanism for opening the channel. We noticed that the mutated residue Arg was facing the channel’s center

Methods

Results

Conclusion