Mechanism of pKID/KIX Association Studied by Molecular Dynamics Free Energy Simulations

The phosphorylated kinase-inducible domain (pKID) associates with the kinase interacting domain (KIX) via a coupled folding and binding mechanism. The pKID domain is intrinsically disordered when unbound and upon phosphorylation at Ser133 binds to the KIX domain adopting a well-defined kinked two-helix structure. In order to identify putative hot spot residues of binding that could serve as an initial stable anchor, we performed in silico alanine scanning free energy simulations. The simulations indicate that charged residues including the phosphorylated central Ser133 of pKID make significant contributions to binding. However, these are of slightly smaller magnitude compared to several hydrophobic side chains not defining a single dominant binding hot spot. Both continuous molecular dynamics (MD) simulations and free energy analysis demonstrate that phosphorylation significantly stabilizes the central kinked motif around Ser133 of pKID and shifts the conformational equilibrium toward the bound conformation already in the absence of KIX. This result supports a view that pKID/KIX association follows in part a conformational selection process. During a 1.5 μs explicit solvent MD simulation, folding of pKID on the surface of KIX was observed after an initial contact at the bound position of the phosphorylation site was enforced following a sequential process of α_<i>A</i> helix association and a stepwise association and folding of the second α_<i>B</i> helix compatible with available experimental results

Molecular Dynamics Analysis of 4E-BP2 Protein Fold Stabilization Induced by Phosphorylation

Protein phosphorylation can affect the interaction with partner proteins but can also induce conformational transitions. In case of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) threonine (Thr) phosphorylation at two turn motifs results in transition from a disordered to a folded structure. In order to elucidate the stabilizing mechanism we employed comparative molecular dynamics (MD) free energy simulations on the turn motifs indicating that Thr-phosphorylation favors a folded whereas dephosphorylation or substitution by Glu residues destabilizes the turn structure. In multiple unrestrained MD simulations at elevated temperature of the 4E-BP2 domain only the double phosphorylated variant remained close to the folded structure in agreement with experiment. Three surface Arg residues were identified as additional key elements for the tertiary structure stabilization of the whole phosphorylated domain. In addition to the local turn structure double phosphorylation also leads to an overall electrostatic stabilization of the folded form compared to wild type and other investigated variants of 4E-BP2. The principles of phosphorylation mediated fold stabilization identified in the present study may also be helpful for identifying other structural motifs that can be affected by phosphorylation or provide a route to design such motifs

Multiscale Simulation of Receptor–Drug Association Kinetics: Application to Neuraminidase Inhibitors

A detailed understanding of the drug–receptor association process is of fundamental importance for drug design. Due to the long time scales of typical binding kinetics, the atomistic simulation of the ligand traveling from bulk solution into the binding site is still computationally challenging. In this work, we apply a multiscale approach of combined Molecular Dynamics (MD) and Brownian Dynamics (BD) simulations to investigate association pathway ensembles for the two prominent H1N1 neuraminidase inhibitors oseltamivir and zanamivir. Including knowledge of the approximate binding site location allows for the selective confinement of detailed but expensive MD simulations and application of less demanding BD simulations for the diffusion controlled part of the association pathway. We evaluate a binding criterion based on the residence time of the inhibitor in the binding pocket and compare it to geometric criteria that require prior knowledge about the binding mechanism. The method ranks the association rates of both inhibitors in qualitative agreement with experiment and yields reasonable absolute values depending, however, on the reaction criteria. The simulated association pathway ensembles reveal that, first, ligands are oriented in the electrostatic field of the receptor. Subsequently, a salt bridge is formed between the inhibitor’s carboxyl group and neuraminidase residue Arg368, followed by adopting the native binding mode. Unexpectedly, despite oseltamivir’s higher overall association rate, the rate into the intermediate salt-bridge state was found to be higher for zanamivir. The present methodology is intrinsically parallelizable and, although computationally demanding, allows systematic binding rate calculation on selected sets of potential drug molecules

Covalent dye attachment influences the dynamics and conformational properties of flexible peptides.

Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the influence on molecular properties when chosing labeling positions for fluorescence experiments

Experimentally obtained FCS curve and model fit function for labeled S-peptide in the presence (black) and absence (gray) of the quenching tryptophan.

<p>Indicated are the four main time regimes of the relevant processes. (I) Photon antibunching: The lifetime of the excited state of the fluorophore determines the shortest temporal separation between two photon emission events. This leads to a decrease in the correlation function for correlation times faster than the lifetime of the excited state. (II) Timescale of the internal conformational dynamics that lead to quenching/unquenching of the fluorophore and dominate the autocorrelation function. These timescales are to be compared with the MD simulations. (III) Photophysical artifacts: Intersystem crossing from the excited singlet state into a dark triplet state with lifetimes in the range of several μs (IV) This correlation regime is dominated by the diffusion of labeled peptides through the confined detection volume of the FCS setup.</p

The RMSD of non-hydrogen (heavy) atoms of residues 1-14 with respect to the unfolded starting structure for simulations with (lower panel) and without (upper panel) labels.

<p>The mean structures of the respectively four largest clusters are shown and their cluster index is indicated (#). Additionally, the simulation time when the clusters mean structures were observed during the simulation is indicated at the bottom right of each cluster structure.</p

The population size of the ten largest clusters in percentage of lifetime compared to the whole trajectory from simulations without (left panel) and with (right panel) fluorescence labels.

<p>Clustering was based on the RMSD and the single linkage algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177139#pone.0177139.ref035" target="_blank">35</a>] with a 0.25 nm cutoff was used.</p

CD spectra of labeled S-peptide with and without Atto655 stacking partner Trp15.

<p>The peak at 220 nm indicates residual <i>α</i>-helix formation and less <i>β</i>-sheet contribution in the labeled S-peptide without Trp15.</p

Atto655/Trp15 fluorescence quenching autocorrelation data fitted with a two-state exponential model function.

<p>Data and fits are shown for MD simulations (A, B) and experimental PET-FCS measurement (C). (A) Data from MD calculated over the whole simulation time (30 μs). (B) Data from MD where the initial 12 μs of the simulation was omitted. (C) Dynamic part of the correlation curve from experimental PET-FCS measurement (red) overlayed with the fitted MD data collected after 12 μs (black).</p

Time evolution of non-hydrogen atoms RMSD of residues 1-14 with respect to the unfolded starting structure and classification into quenched and fluorescent states in the time frame between 27.5–28.0 μs.

<p>Although the backbone was mostly locked in cluster #1 conformation during this time span, spontaneous unstacking of Atto655/Trp15 was observed. Two exemplary structures shortly before and after an unstacking event are shown below. The stacked (grey) configuration quenches the Atto655 fluorescence, while the unstacked (red) configuration allows fluorescence. Unstacking was observed to occur on the subnanosecond timescale and is not coupled to a noticable change of the overall RMSD in this cluster.</p