Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.

Vanessa Leone,Christine Ziegler,Burkhard Endeward,Monika Sharma,Thomas F Prisner,Katharina Kossmann,Caroline Koshy,Lucy R Forrest,Izabela Waclawska

doi:10.1085/jgp.201812111

Abstract

Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.

Highlights

Molecular mechanisms of signaling, solute transport, and permeation across membranes typically require a membrane protein to undergo one or more conformational changes
Site-directed spin labeling of betaine permease (BetP) Nitroxide radicals were introduced into the cysteine-less BetP mutant C252T (Rübenhagen et al, 2000; Ott et al, 2008) by sitedirected spin labeling
MTS spin labels were covalently linked to these residues with excellent efficiency (112 ± 22.4%), and the labeled construct was capable of sodium-dependent [14C]-betaine uptake when reconstituted into liposomes (Fig. 2 A)

Summary

Introduction

Solute transport, and permeation across membranes typically require a membrane protein to undergo one or more conformational changes. Coupling the conformational changes to binding of sodium, for example, moving along a preexisting concentration gradient, energizes the transport process, resulting in net accumulation of the substrate. Substrates, and other environmental factors such as the lipid composition can all affect these conformational equilibria. Understanding how these factors act on membrane proteins during alternating access requires an atomistic description of the conformational ensembles; only can the underlying energy landscapes, and the shifts therein, be accurately described (Faraldo-Gómez and Forrest, 2011; Masureel et al, 2014; Liao et al, 2016; Ruan et al, 2017). A number of studies have attempted to extend the interpretations from these discrete snapshots into ensemble descriptions reflecting more native

Methods

Results

Conclusion