Structural Refinement of Proteins by Restrained Molecular Dynamics Simulations with Non-interacting Molecular Fragments

<div><p>The knowledge of multiple conformational states is a prerequisite to understand the function of membrane transport proteins. Unfortunately, the determination of detailed atomic structures for all these functionally important conformational states with conventional high-resolution approaches is often difficult and unsuccessful. In some cases, biophysical and biochemical approaches can provide important complementary structural information that can be exploited with the help of advanced computational methods to derive structural models of specific conformational states. In particular, functional and spectroscopic measurements in combination with site-directed mutations constitute one important source of information to obtain these mixed-resolution structural models. A very common problem with this strategy, however, is the difficulty to simultaneously integrate all the information from multiple independent experiments involving different mutations or chemical labels to derive a unique structural model consistent with the data. To resolve this issue, a novel restrained molecular dynamics structural refinement method is developed to simultaneously incorporate multiple experimentally determined constraints (e.g., engineered metal bridges or spin-labels), each treated as an individual molecular fragment with all atomic details. The internal structure of each of the molecular fragments is treated realistically, while there is no interaction between different molecular fragments to avoid unphysical steric clashes. The information from all the molecular fragments is exploited simultaneously to constrain the backbone to refine a three-dimensional model of the conformational state of the protein. The method is illustrated by refining the structure of the voltage-sensing domain (VSD) of the Kv1.2 potassium channel in the resting state and by exploring the distance histograms between spin-labels attached to T4 lysozyme. The resulting VSD structures are in good agreement with the consensus model of the resting state VSD and the spin-spin distance histograms from ESR/DEER experiments on T4 lysozyme are accurately reproduced.</p></div

Details of the models of the VSD of the Kv1.2 channel.

<p>Details of the models of the VSD of the Kv1.2 channel.</p

Final configurations of the four models of the VSD with different molecular fragments.

<p>The final configuration (left panel) and the correlation between the C_<i>β</i>-C_<i>β</i> distance and the S_<i>γ</i>-Cd²⁺, O_<i>δ2</i>-Mg²⁺, N_<i>ε</i>-Zn²⁺, or N_<i>ζ</i>-O_<i>ε2</i> distance in each molecular fragment (right panel) of (A) model-1, (B) model-2, (C) model-3, and (D) model-4. The last 10 ns trajectories of the restrained MD simulations were used to calculate the average values and standard deviations of the distances.</p

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The backbone RMSD values of the TM helices S1–S4 between different models.

<p>The backbone RMSD values of the TM helices S1–S4 between different models.</p

Schematic of the molecular fragments method.

<p>(A) Each structural constraint (e.g., a metal ion bridge or a spin-label) is present as a molecular fragment with all atomic details in the system. (B) The residue(s) in the molecular fragment (colored in red) is/are attached to the targeting residue(s) in the wild-type protein, via harmonic restraints, with their backbone atoms (N, C, O and C_<i>α</i>) staying on top of each other, respectively. The residue in the molecular fragment does not have interactions with its targeting residue in the wild-type protein (residue <i>i</i>), and the interactions between the backbone atoms of the residue in the molecular fragment and the backbone atoms of the two nearby residues in the wild-type protein (residues <i>i</i>-1 and <i>i</i>+1; embraced by red dashed lines) are also turned off. (C) During the restrained MD simulations, the interactions within each molecular fragment are evaluated accurately triggering the conformational changes of the wild-type protein, while different molecular fragments do not have interactions with one another.</p

The final coordinates of the molecular fragments in model-3 after the restrained MD simulation.

<p>(A–F) The configurations of the four TM helices before and after the restrained MD simulation are shown in transparent and solid ribbons, respectively.</p

Configurations of the spin-labels attached to T4 lysozyme.

<p>(A) A snapshot of the T4 lysozyme system with 34 MTSSL spin-labels. The spin-label at each site has 25 copies and highlighted in the ball-and-stick representation. (B) Distance histograms of four pairs of spin-labels from ESR/DEER experiments (black solid line), and the molecular dynamics simulations without (blue dashed line) and with (red dashed line) an energy restraint. Please see the Supplementary Material, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004368#pcbi.1004368.s003" target="_blank">S3</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004368#pcbi.1004368.s004" target="_blank">S4</a> Figs for the other 47 distance distributions.</p

Superimposition of the initial and final configurations of the four models of the VSD.

<p>(A) model-1, (B) model-2, (C) model-3, and (D) model-4. The initial and final configurations of the backbone atoms are represented in transparent and solid sticks, respectively.</p

Starting configurations of the four models of the VSD with different molecular fragments.

<p>(A) model-1. (B) model-2. (C) model-3. (D) model-4. The four TM helices, S1 (gray), S2 (yellow), S3 (red) and S4 (blue), are represented in transparent ribbons. Molecular fragments, I177C–Cd²⁺–R294C, I230C–Cd²⁺–R294C, I230D–Mg²⁺–F267D, F233W/E236–R294K, I230H–Zn²⁺–A291H, I268C–Cd²⁺–A287C, and T269C–Cd²⁺–A291C, are colored in red, yellow, green, magenta, cyan, orange, and blue, respectively, with amino acids being represented in sticks and ions in spheres.</p