Abstract
The structural dynamics governing collective motions in oligomeric membrane proteins play key roles in vital biomolecular processes at cellular membranes. In this study, we present a structural refinement approach that combines solid-state NMR experiments and molecular simulations to accurately describe concerted conformational transitions identifying the overall structural, dynamical, and topological states of oligomeric membrane proteins. The accuracy of the structural ensembles generated with this method is shown to reach the statistical error limit, and is further demonstrated by correctly reproducing orthogonal NMR data. We demonstrate the accuracy of this approach by characterising the pentameric state of phospholamban, a key player in the regulation of calcium uptake in the sarcoplasmic reticulum, and by probing its dynamical activation upon phosphorylation. Our results underline the importance of using an ensemble approach to characterise the conformational transitions that are often responsible for the biological function of oligomeric membrane protein states.
Highlights
In the present study, we present an optimal approach to characterise in detail the structure and dynamics of oligomeric membrane proteins by using oriented solid-state nuclear magnetic resonance experiments in combination with restrained ensemble-averaged molecular dynamics (MD) simulations[23,24,25]
This calibration was performed thorough the ‘reference ensemble’, which has been extensively described in the literature[27,28] (Supplementary Material). This in silico experiment identified the optimal averaging scheme to be composed of 16 replicas of the system in combination with internal averaging (Fig. 1). We demonstrate that this approach can generate structural ensembles with an error reaching the statistical limit (Supplementary Methods and Fig. S1), which provides an estimate of the significant accuracy by which chemical shift anisotropy (CSA) and dipolar couplings (DC) restrained MD are able to refine structure, dynamics and topology of oligomeric MPs
It is crucially important to characterise the conformational transitions in oligomeric membrane proteins, as these proteins often exert their biological function via collective motions that are actively employed in processes such as signal transduction or molecular transport across the membrane
Summary
We present an optimal approach to characterise in detail the structure and dynamics of oligomeric membrane proteins by using oriented solid-state nuclear magnetic resonance (ssNMR) experiments in combination with restrained ensemble-averaged molecular dynamics (MD) simulations[23,24,25]. When treating experimental data of oligomeric MPs, the complex averaging of the NMR observables poses significant challenges. We show that ensemble-average MD can be used to successfully characterise structure and dynamics of the pentameric state of PLN in both phosphorylated (pS16) and non-phosphorylated forms. The structural ensembles resulting from this study describe accurately the nature of the dynamic activation of the pentameric PLN upon phosphorylation, which is an underlying mechanism of SERCA regulation
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