Structural Ensembles of Intrinsically Disordered Proteins using Molecular Dynamics Simulation

Structural Ensembles of Intrinsically Disordered Proteins Depend Strongly on Force Field

Molecular dynamics (MD) simulations of six upgraded empirical force fields were compared and evaluated with short peptides, intrinsically disordered proteins, and folded proteins using trajectories of 1, 1.5, 5, or 10 μs (five replicates of 200 ns, 300 ns, 1 μs, or 2 μs) for each system. Previous studies have shown that different force fields, water models, simulation methods, and parameters can affect simulation outcomes. Here, the MD simulations were done in an explicit solvent with RS-peptide, HEWL19, HIV-rev, β amyloid (Aβ)-40, Aβ-42, phosphodiesterase-γ, CspTm, and ubiquitin using ff99IDPs, ff14IDPs, ff14IDPSFF, ff03w, CHARMM36m, and CHARMM22* force fields. The IDP ensembles generated by six all-atom empirical force fields were compared against NMR data. Despite using identical starting structures and simulation parameters, ensembles obtained with different force fields exhibit significant differences in NMR RMDs, secondary structure contents, and global properties such as the radius of gyration. The intrinsically disordered protein (IDP)-specific force fields could substantially reproduce the experimental observables in force field comparison: they have the lowest error in chemical shifts and J-couplings for short peptides/proteins, reasonably well for large IDPs and reasonably well with the radius of gyration. A high population of disorderness was observed in the IDP-specific force field for the IDP ensemble with a fraction of β sheets for β-amyloids. CHARMM22* performs better for many observables; however, it still has a preference toward the helicity for short peptides. The results of β-amyloid 42 starting from two different initial structures (Aβ421Z0Q and Aβ42model) were also compared with DSSP and NMR data. The results obtained with IDP-specific force fields within 2 μs simulation time are similar, even though starting from different structures. The current force fields perform equally well for folded proteins. The results of currently developed or modified force fields for IDPs are capable of enlightening the overall performance of the force field for disordered as well as folded proteins, thereby contributing to force field development.

Proteins with intrinsically disordered regions (IDRs) have large hydrodynamic radii, compared with globular proteins of equivalent weight. Recent experiments showed that IDRs with large radii can create steric pressure to drive membrane curvature during Clathrin-mediated endocytosis (CME). Epsin and Eps15 are two CME proteins with IDRs that contain multiple motifs for binding the adaptor protein AP2, but the impact of AP2-binding on these IDRs is unknown. Some IDRs acquire binding-induced function by forming a folded quaternary structure, but we hypothesize that the IDRs of Epsin and/or Eps15 acquire binding-induced function by increasing their steric volume. We explore this hypothesis in silico by generating conformational ensembles of the IDRs of Epsin (4 million structures) or Eps15 (3 million structures), then estimating the impact of AP2-binding on Radius of Gyration (RG). Results show that the ensemble of Epsin IDR conformations that accommodate AP2 binding has a right-shifted distribution of RG (larger radii) than the unbound Epsin ensemble. In contrast, the ensemble of Eps15 IDR conformations has comparable RG distribution between AP2-bound and unbound. We speculate that AP2 triggers the Epsin IDR to function through binding-induced-expansion, which could increase steric pressure and membrane bending during CME.

Developing a Protocol for Ensemble and Vibrational Probe-Containing Molecular Dynamics Simulations of the Nipah Ntail-XD Complex

Intrinsically disordered regions (IDRs) or proteins (IDPs), which play crucial biological functions in essential biological processes of life, do not have well-defined secondary or tertiary structures when isolated in solution. The highly dynamic properties and conformational heterogeneity of IDPs make them challenging to study with traditional experimental techniques. As a powerful complementary tool for experiments, all-atom molecular dynamics simulation can obtain detailed conformational information on IDPs, but the limitation of force field accuracy is a challenge for reproducing IDP conformers. Here, we compared five empirical all-atom force fields AMBER03, AMBER99SB-ILDN, CHARMM27, OPLS-AA/L and CHARMM36m in modeling the conformational ensembles of wild-type peptide TAD2(41-62) from the human p53 tumor suppressor. Our results show that for the model peptide, the newest force field CHARMM36m produces more expanded coil ensemble followed by AMBER99SB-ILDN; CHARMM27 displays a predominant propensity for a helical structure; whereas OPLS-AA/L exhibits a apparent preference for a β-sheet structure and yields the most compact conformation. In the comparison of the simulated dimensions with theoretical prediction and the back-calculated chemical shifts with experimental measurements, AMBER99SB-ILDN gives a more consistent agreement than the other force fields. In addition, the region from residues 47 to 55, which commonly forms an amphipathic α-helix upon binding target proteins according to experimental observation, could form a helical structure with a different probability population in our simulations with different force fields. This implies that the binding process might be conducted by, or partly by "conformation selection" for this peptide. This work indicates that force field development for modeling general IDPs accurately has a long way to go, and more detailed experimental data of IDPs are also in demand.

Improved Accuracy and Convergence of Intrinsically Disordered Protein Molecular Dynamics Simulations Using the ff14IDPSFF Force Field

Intrinsically disordered proteins (IDPs) are highly dynamic systems that play an important role in cell signaling processes and their misfunction often causes human disease. Proper understanding of IDP function not only requires the realistic characterization of their three-dimensional conformational ensembles at atomic-level resolution but also of the time scales of interconversion between their conformational substates. Large sets of experimental data are often used in combination with molecular modeling to restrain or bias models to improve agreement with experiment. It is shown here for the N-terminal transactivation domain of p53 (p53TAD) and Pup, which are two IDPs that fold upon binding to their targets, how the latest advancements in molecular dynamics (MD) simulations methodology produces native conformational ensembles by combining replica exchange with series of microsecond MD simulations. They closely reproduce experimental data at the global conformational ensemble level, in terms of the distribution properties of the radius of gyration tensor, and at the local level, in terms of NMR properties including 15N spin relaxation, without the need for reweighting. Further inspection revealed that 10–20% of the individual MD trajectories display the formation of secondary structures not observed in the experimental NMR data. The IDP ensembles were analyzed by graph theory to identify dominant inter-residue contact clusters and characteristic amino-acid contact propensities. These findings indicate that modern MD force fields with residue-specific backbone potentials can produce highly realistic IDP ensembles sampling a hierarchy of nano- and picosecond time scales providing new insights into their biological function.

Molecular simulations of IDPs: From ensemble generation to IDP interactions leading to disorder-to-order transitions.

Measuring Disorder and Dynamical Properties of FG-Nucleoporins

The identification and characterization of the main conformations from a protein population are a challenging and inherently high-dimensional problem. Here, we evaluate the performance of the Secondary sTructural Ensembles with machine LeArning (StELa) double-clustering method, which clusters protein structures based on the relationship between the φ and ψ dihedral angles in a protein backbone and the secondary structure of the protein, thus focusing on the local properties of protein structures. The classification of states as vectors composed of the clusters' indices arising naturally from the Ramachandran plot is followed by the hierarchical clustering of the vectors to allow for the identification of the main features of the corresponding free energy landscape (FEL). We compare the performance of StELa with the established root-mean-squared-deviation (RMSD)-based clustering algorithm, which focuses on global properties of protein structures and with Combinatorial Averaged Transient Structure (CATS), the combinatorial averaged transient structure clustering method based on distributions of the φ and ψ dihedral angle coordinates. Using ensembles of conformations from molecular dynamics simulations of intrinsically disordered proteins (IDPs) of various lengths (tau protein fragments) or short fragments from a globular protein, we show that StELa is the clustering method that identifies many of the minima and relevant energy states around the minima from the corresponding FELs. In contrast, the RMSD-based algorithm yields a large number of clusters that usually cover most of the FEL, thus being unable to distinguish between states, while CATS does not sample well the FELs for long IDPs and fragments from globular proteins.

Phosphorylation is a major post-translational modification that is involved in the regulation of the dynamics and function of intrinsically disordered proteins (IDPs). We recently characterized a phenomenon, which we termed n-phosphate collaborations (nP-collabs), where bulk cations form stable bridges between several phosphoresidues in all-atom molecular dynamic simulations. nP-collabs were found to be sensitive to the combination of force fields and cation types. Here, we attempt to assess the physical relevance of these nP-collabs by evaluating the strength of the cation/phosphate interaction through osmotic coefficient (ϕ) calculations on the model 2Na+HPO42- and 2K+HPO42- salts, using different classical force fields for phosphorylations. All force fields were found to overestimate the strength of the interaction to various degrees. We thus designed new parameters for CHARMM36m and AmberFF99SB-ILDN using the Electronic Continuum Correction (ECC) approach, which provide remarkable agreement for ϕ values for both cation types and over a range of concentrations. We provide a preliminary test of these ECC parameters for phosphorylations by simulating the 7-fold-phosphorylated rhodopsin peptide 7PP and comparing secondary chemical shifts to experimental data. Conformational ensembles resulting from the ECC-derived phosphorylated force fields display both qualitative and quantitative improvements with regard to full-charge force fields. We thus conclude that long-lasting nP-collabs are artifacts for classical force fields born from the lack of explicit polarization, and propose a possible computational strategy for the extensive parametrization of phosphorylations. The presence of long-lived nP-collabs in simulations produced using classical force fields is therefore a serious concern for the accurate modeling of multiphosphorylated peptides and IDPs, which are at the center of research questions regarding neurodegenerative diseases such as Alzheimer's or Parkinson's.

Transcription factor cyclic Adenosine monophosphate response-element binding protein plays a critical role in the cyclic AMP response pathway via its intrinsically disordered kinase inducible transactivation domain (KID). KID is one of the most studied intrinsically disordered proteins (IDPs), although most previous studies focus on characterizing its disordered state structures. An interesting question that remains to be answered is how the order-disorder transition occurs at experimental conditions. Thanks to the newly developed IDP-specific force field ff14IDPSFF, the quality of conformer sampling for IDPs has been dramatically improved. In this study, molecular dynamics (MD) simulations were used to study the order-to-disorder transition kinetics of KID based on the good agreement with the experiment on its disordered-state properties. Specifically, we tested four force fields, ff99SBildn, ff99IDPs, ff14IDPSFF, and ff14IDPs in the simulations of KID and found that ff14IDPSFF can generate more diversified disordered conformers and also reproduce more accurate experimental secondary chemical shifts. Kinetics analysis of MD simulations demonstrates that the order-disorder transition of KID obeys the first-order kinetics, and the transition nucleus is I127/L128/L141. The possible transition pathways from the nucleus to the last folded residues were identified as I127-R125-L138-L141-S143-A145 and L128-R125-L138-L141-S143-A145 based on a residue-level dynamical network analysis. These computational studies not only provide testable prediction/hypothesis on the order-disorder transition of KID but also confirm that the ff14IDPSFF force field can be used to explore the correlation between the structure and function of IDPs.

The sensitivity of molecular dynamics simulations to variations in the force field has been examined in relation to a set of 36 structures corresponding to 31 proteins simulated by using different versions of the GROMOS force field. The three parameter sets used (43a1, 53a5, and 53a6) differ significantly in regard to the nonbonded parameters for polar functional groups and their ability to reproduce the correct solvation and partitioning behavior of small molecular analogues of the amino acid side chains. Despite the differences in the force field parameters no major differences could be detected in a wide range of structural properties such as the root-mean-square deviation from the experimental structure, radii of gyration, solvent accessible surface, secondary structure, or hydrogen bond propensities on a 5 to 10 ns time scale. The small differences that were observed correlated primarily with the presence of charged residues as opposed to residues that differed most between the parameter sets. The work highlights the variation that can be observed in nanosecond simulations of protein systems and implications of this for force field validation, as well as for the analysis of protein simulations in general.

Salts modulate the behavior of intrinsically disordered proteins (IDPs) and influence the formation of membraneless organelles through liquid-liquid phase separation (LLPS). In low ionic strength solutions, IDP conformations are perturbed by the screening of electrostatic interactions, independent of the salt identity. In this regime, insight into the IDP behavior can be obtained using the theory for salt-induced transitions in charged polymers. However, salt-specific interactions with the charged and uncharged residues, known as the Hofmeister effect, influence IDP behavior in high ionic strength solutions. There is a lack of reliable theoretical models in high salt concentration regimes to predict the salt effect on IDPs. We propose a simulation methodology using a coarse-grained IDP model and experimentally measured water to salt solution transfer free energies of various chemical groups that allowed us to study the salt-specific transitions induced in the IDPs conformational ensemble. We probed the effect of three different monovalent salts on five IDPs belonging to various polymer classes based on charged residue content. We demonstrate that all of the IDPs of different polymer classes behave as self-avoiding walks (SAWs) at physiological salt concentration. In high salt concentrations, the transitions observed in the IDP conformational ensembles are dependent on the salt used and the IDP sequence and composition. Changing the anion with the cation fixed can result in the IDP transition from a SAW-like behavior to a collapsed globule. An important implication of these results is that a suitable salt can be identified to induce condensation of an IDP through LLPS.

Over 40% of eukaryotic proteomic sequences have been predicted to be intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) and confirmed to be associated with many diseases. However, widely used force fields cannot well reproduce the conformers of IDPs. Previously the ff99IDPs force field was released to simulate IDPs with CMAP energy corrections for the eight disorder-promoting residues. In order to further confirm the performance of ff99IDPs, three representative IDP systems (arginine-rich HIV-1 Rev, aspartic proteinase inhibitor IA3, and α-synuclein) were used to test and evaluate the simulation results. The results show that for free disordered proteins, the chemical shifts from the ff99IDPs simulations are in quantitative agreement with those from reported NMR measurements and better than those from ff99SBildn. Thus, ff99IDPs can sample more clusters of disordered conformers than ff99SBildn. For structural proteins, both ff99IDPs and ff99SBildn can well reproduce the conformations. In general, ff99IDPs can successfully be used to simulate the conformations of IDPs and IDRs in both bound and free states. However, relative errors could still be found at the boundaries of ordered residues scattered in long disorder-promoting sequences. Therefore, polarizable force fields might be one of the possible ways to further improve the performance on IDPs.