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Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1

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Abstract

The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16–31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 ϕ torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular α-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured 3J(HN-H)-coupling constants, indicating that different conformers of GCN4p16–31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and 3J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and 3J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16–31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16–31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.

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Acknowledgments

Financial support by the National Centre of Competence in Research (NCCR) in structural biology and by grant number 200020-121913 of the Swiss National Science Foundation (SNSF) and by grant number 228076 of the European Research Council (ERC) to W. F. van G., and by the Slovenian Research Agency (ARRS), grant number Z1-9576 to J. D., is gratefully acknowledged. We would like to thank Jane R. Allison for help with the local-elevation biased 3J-coupling restraining, and Andrei Alexandrescu and Wolfgang Jahnke for their constructive criticism of the manuscript.

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Correspondence to Wilfred F. van Gunsteren.

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Dolenc, J., Missimer, J.H., Steinmetz, M.O. et al. Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1. J Biomol NMR 47, 221–235 (2010). https://doi.org/10.1007/s10858-010-9425-9

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