Toward a predictive understanding of slow methyl group dynamics in proteins

The development of the most recent generation of molecular mechanics force fields promises an increasingly predictive understanding of the protein dynamics-function relationship. Based on extensive validation against various types of experimental data, the AMBER force field ff99SB was benchmarked in recent years as a favorable force field for protein simulations. Recent improvements of the side chain and backbone potentials, made by different groups, led to the ff99SB-ILDN and ff99SBnmr1 force fields, respectively. The combination of these potentials into a unified force field, termed ff99SBnmr1-ILDN, was used in this study to perform a microsecond time scale molecular dynamics simulation of free ubiquitin in explicit solvent for validation against an extensive set of experimental NMR methyl group residual dipolar couplings. Our results show a high level of consistency between the experimental data and the values predicted from the molecular dynamics trajectory reflecting a systematically improved performance of ff99SBnmr1-ILDN over the original ff99SB force field. Moreover, the unconstrained ff99SBnmr1-ILDN MD ensemble achieves a similar level of agreement as the recently introduced EROS ensemble, which was constructed based on a large body of NMR data as constraints, including the methyl residual dipolar couplings. This suggests that ff99SBnmr1-ILDN provides a high-quality representation of the motions of methyl-bearing protein side chains, which are sensitive probes of protein-protein and protein-ligand interactions.     

Improvements of the hierarchical approach for predicting RNA tertiary structure

Computational prediction of RNA tertiary structures is a significant challenge, especially for longer RNA and pseudoknots. At present it is still difficult to do this by pure all-atom molecular dynamics simulation. One of possible approaches is through hierarchical steps: from sequence to secondary structure and then to tertiary structure. Here we present improvements of two key steps of this approach, the manual adjustment of atom clashes and bond stretches and molecular dynamics refinement. We provide an energy function to find the locations of atom clashes and bond stretches and to guide their manual adjustment and a new scheme of molecular dynamics refinement using a tested combination of solvent model and the ff98 Amber force field suitable for RNA. We predicted with higher accuracy the tertiary structures of nine typical RNA molecules of lengths from 12 to 52, including hairpins, duplex helices and pseudoknots.     

Interchain hydrophobic clustering promotes rigidity in HIV-1 protease flap dynamics: new insights from Molecular Dynamics

The dynamics of HIV-1 protease (HIV-pr), a drug target for HIV infection, has been studied extensively by both computational and experimental methods. The flap dynamics of HIV-pr is considered to be more important for better ligand binding and enzymatic actions. Moreover, it has been demonstrated that the drug-induced mutations can change the flap dynamics of HIV-pr affecting the binding affinity of the ligands. Therefore, detailed understanding of flap dynamics is essential for designing better inhibitors. Previous computational investigations observed significant variation in the flap opening in nanosecond time scale indicating that the dynamics is highly sensitive to the simulation protocols. To understand the sensitivity of the flap dynamics on the force field and simulation protocol, molecular dynamics simulations of HIV-pr have been performed with two different AMBER force fields, ff99 and ff02. Two different trajectories (20?ns each) were obtained using the ff99 and ff02 force field. The results showed polarizable force field (ff02) make the flap tighter than the nonpolarizable force field (ff99). Some polar interactions and hydrogen bonds involving flap residues were found to be stronger with ff02 force field. The formation of interchain hydrophobic cluster (between flap tip of one chain and active site wall of another chain) was found to be dominant in the semi-open structures obtained from the simulations irrespective of the force field. It is proposed that an inhibitor, which will promote this interchain hydrophobic clustering, may make the flaps more rigid, and presumably the effect of mutation would be small on ligand binding.     

Improved side‐chain torsion potentials for the Amber ff99SB protein force field

Recent advances in hardware and software have enabled increasingly long molecular dynamics (MD) simulations of biomolecules, exposing certain limitations in the accuracy of the force fields used for such simulations and spurring efforts to refine these force fields. Recent modifications to the Amber and CHARMM protein force fields, for example, have improved the backbone torsion potentials, remedying deficiencies in earlier versions. Here, we further advance simulation accuracy by improving the amino acid side‐chain torsion potentials of the Amber ff99SB force field. First, we used simulations of model alpha‐helical systems to identify the four residue types whose rotamer distribution differed the most from expectations based on Protein Data Bank statistics. Second, we optimized the side‐chain torsion potentials of these residues to match new, high‐level quantum‐mechanical calculations. Finally, we used microsecond‐timescale MD simulations in explicit solvent to validate the resulting force field against a large set of experimental NMR measurements that directly probe side‐chain conformations. The new force field, which we have termed Amber ff99SB‐ILDN, exhibits considerably better agreement with the NMR data. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.     

The ABCs of molecular dynamics simulations on B-DNA, circa 2012

This article provides a retrospective on the ABC initiative in the area of all-atom molecular dynamics (MD) simulations including explicit solvent on all tetranucleotide steps of duplex B-form DNA duplex, ca. 2012. The ABC consortium has completed two phases of simulations, the most current being a set of 50-100 trajectories based on the AMBER ff99 force field together with the parmbsc0 modification. Some general perspectives on the field of MD on DNA and sequence effects on DNA structure are provided, followed by an overview our MD results, including a detailed comparison of the ff99/parmbsc0 results with crystal and NMR structures available for d(CGCGAATTCGCG). Some projects inspired by or related to the ABC initiative and database are also reviewed, including methods for the trajectory analyses, informatics of dealing with the large database of results, compressions of trajectories for efficacy of distribution, DNA solvation by water and ions, parameterization of coarse-grained models with applications and gene finding and genome annotation.     

On the origins of the weak folding cooperativity of a designed ββα ultrafast protein FSD-1

FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96) coupled with the implicit solvent model (IGB = 5), we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K) of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K) as well as the denatured structures.     

Force field influences in beta-hairpin folding simulations

All-atom force fields are now routinely used for more detailed understanding of protein folding mechanisms. However, it has been pointed out that use of all-atom force fields does not guarantee more accurate representations of proteins; in fact, sometimes it even leads to biased structural distributions. Indeed, several issues remain to be solved in force field developments, such as accurate treatment of implicit solvation for efficient conformational sampling and proper treatment of backbone interactions for secondary structure propensities. In this study, we first investigate the quality of several recently improved backbone interaction schemes in AMBER for folding simulations of a beta-hairpin peptide, and further study their influences on the peptide's folding mechanism. Due to the significant number of simulations needed for a thorough analysis of tested force fields, the implicit Poisson-Boltzmann solvent was used in all simulations. The chosen implicit solvent was found to be reasonable for studies of secondary structures based on a set of simulations of both alpha-helical and beta-hairpin peptides with the TIP3P explicit solvent as benchmark. Replica exchange molecular dynamics was also utilized for further efficient conformational sampling. Among the tested AMBER force fields, ff03 and a revised ff99 force field were found to produce structural and thermodynamic data in comparably good agreement with the experiment. However, detailed folding pathways, such as the order of backbone hydrogen bond zipping and the existence of intermediate states, are different between the two force fields, leading to force field-dependent folding mechanisms.     

Why the OPLS-AA force field cannot produce the β-hairpin structure of H1 peptide in solution when comparing with the GROMOS 43A1 force field?

The optimal combination of force field and water model is an essential problem that is able to increase molecular dynamics simulation quality for different types of proteins and peptides. In this work, an attempt has been made to explore the problem by studying H1 peptide using four different models based on different force fields, water models and electrostatic schemes. The driving force for H1 peptide conformation transition and the reason why the OPLS-AA force field cannot produce the β-hairpin structure of H1 peptide in solution while the GROMOS 43A1 force field can do were investigated by temperature replica exchange molecular dynamics simulation (T-REMD). The simulation using the GROMOS 43A1 force field preferred to adopt a β-hairpin structure, which was in good agreement with the several other simulations and the experimental evidences. However, the simulation using the OPLS-AA force field has a significant difference from the simulations with the GROMOS 43A1 force field simulation. The results show that the driving force in H1 peptide conformation transition is solvent exposure of its hydrophobic residues. However, the subtle balances between residue-residue interactions and residue-solvent interaction are disrupted by using the OPLS-AA force field, which induced the reduction in the number of residue-residue contact. Similar solvent exposure of the hydrophobic residues is observed for all the conformations sampled using the OPLS-AA force field. For H1 peptide which exhibits large solvent exposure of the hydrophobic residues, the GROMOS 43A1 force field with the SPC water model can provide more accurate results.     

Evaluating amber force fields using computed NMR chemical shifts

NMR chemical shifts can be computed from molecular dynamics (MD) simulations using a template matching approach and a library of conformers containing chemical shifts generated from ab initio quantum calculations. This approach has potential utility for evaluating the force fields that underlie these simulations. Imperfections in force fields generate flawed atomic coordinates. Chemical shifts obtained from flawed coordinates have errors that can be traced back to these imperfections. We use this approach to evaluate a series of AMBER force fields that have been refined over the course of two decades (ff94, ff96, ff99SB, ff14SB, ff14ipq, and ff15ipq). For each force field a series of MD simulations are carried out for eight model proteins. The calculated chemical shifts for the ¹H, ¹⁵N, and ¹³C^a atoms are compared with experimental values. Initial evaluations are based on root mean squared (RMS) errors at the protein level. These results are further refined based on secondary structure and the types of atoms involved in nonbonded interactions. The best chemical shift for identifying force field differences is the shift associated with peptide protons. Examination of the model proteins on a residue by residue basis reveals that force field performance is highly dependent on residue position. Examination of the time course of nonbonded interactions at these sites provides explanations for chemical shift differences at the atomic coordinate level. Results show that the newer ff14ipq and ff15ipq force fields developed with the implicitly polarized charge method perform better than the older force fields.     

Microsecond simulations of mdm2 and its complex with p53 yield insight into force field accuracy and conformational dynamics

The p53‐MDM2 complex is both a major target for cancer drug development and a valuable model system for computational predictions of protein‐ligand binding. To investigate the accuracy of molecular simulations of MDM2 and its complex with p53, we performed a number of long (200 ns to 1 µs) explicit‐solvent simulations using a range of force fields. We systematically compared nine popular force fields (AMBER ff03, ff12sb, ff14sb, ff99sb, ff99sb‐ildn, ff99sb‐ildn‐nmr, ff99sb‐ildn‐phi, CHARMM22*, and CHARMM36) against experimental chemical shift data, and found similarly accurate results, with microsecond simulations achieving better agreement compared to 200‐ns trajectories. Although the experimentally determined apo structure has a closed binding cleft, simulations in all force fields suggest the apo state of MDM2 is highly flexible, and able to sample holo‐like conformations, consistent with a conformational selection model. Initial structuring of the MDM2 lid region, known to competitively bind the binding cleft, is also observed in long simulations. Taken together, these results show molecular simulations can accurately sample conformations relevant for ligand binding. We expect this study to inform future computational work on folding and binding of MDM2 ligands. Proteins 2015; 83:1665–1676. © 2015 Wiley Periodicals, Inc.     

Flexibility of the Bacterial Chaperone Trigger Factor in Microsecond-Timescale Molecular Dynamics Simulations

The bacterial chaperone trigger factor (TF) is the first chaperone to be encountered by a nascent protein chain as it emerges from the ribosome exit tunnel. Experimental results suggest that TF possesses considerable conformational flexibility, and in an attempt to provide an atomic-level view of this flexibility, we have performed independent 1.5-μs molecular dynamics simulations of TF in explicit solvent using two different simulation force fields (OPLS-AA/L and AMBER ff99SB-ILDN). Both simulations indicate that TF possesses tremendous flexibility, with huge excursions from the crystallographic conformation caused by reorientations of the protein’s constituent domains; both simulations also predict the formation of extensive contacts between TF’s PPIase domain and the Arm 1 domain that is involved in nascent-chain binding. In the OPLS simulation, however, TF rapidly settles into a very compact conformation that persists for at least 1 μs, whereas in the AMBER simulation, it remains highly dynamic; additional simulations in which the two force fields were swapped suggest that these differences are at least partly attributable to sampling issues. The simulation results provide potential rationalizations of a number of experimental observations regarding TF’s conformational behavior and have implications for using simulations to model TF’s function on translating ribosomes.     

Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non-fluorinated parallel dimeric coiled-coils

The alpha-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of the coiled-coil motifs, including those modified by fluorination, several fluorinated and nonfluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiled-coil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER used a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, and ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly, better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller L-ornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled-coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine when compared with 5,5,5,5',5',5'-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5',5',5'-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may provide a driving force for coiled-coil dimer formation. Estimation of the ellipticity at 222 nm from a series of snapshots from the MD simulations with DicroCalc shows distinct increases in the ellipticity when the coiled-coil is fluorinated, which suggests that the helicity in the folded coiled-coils is greater when fluorinated.     

Exploring protein native states and large-scale conformational changes with a modified generalized born model

Implicit solvation models provide, for many applications, a reasonably accurate and computationally effective way to describe the electrostatics of aqueous solvation. Here, a popular analytical Generalized Born (GB) solvation model is modified to improve its accuracy in calculating the solvent polarization part of free energy changes in large-scale conformational transitions, such as protein folding. In contrast to an earlier GB model (implemented in the AMBER-6 program), the improved version does not overstabilize the native structures relative to the finite-difference Poisson-Boltzmann continuum treatment. In addition to improving the energy balance between folded and unfolded conformers, the algorithm (available in the AMBER-7 and NAB molecular modeling packages) is shown to perform well in more than 50 ns of native-state molecular dynamics (MD) simulations of thioredoxin, protein-A, and ubiquitin, as well as in a simulation of Barnase/Barstar complex formation. For thioredoxin, various combinations of input parameters have been explored, such as the underlying gas-phase force fields and the atomic radii. The best performance is achieved with a previously proposed modification to the torsional potential in the Amber ff99 force field, which yields stable native trajectories for all of the tested proteins, with backbone root-mean-square deviations from the native structures being approximately 1.5 A after 6 ns of simulation time. The structure of Barnase/Barstar complex is regenerated, starting from an unbound state, to within 1.9 A relative to the crystal structure of the complex.     

Exploring potentially alternative non-canonical DNA duplex structures through simulation

Hopkins proposed an alternative and chirally distinct family of double-stranded DNA (dsDNA) models that have antiparallel chains with 5′→3′ senses opposite to those of the right-handed Watson–Crick (WC) family. Termed configuration II, this family of dsDNA models contains both right-handed (II-R) and left-handed (II-L) forms, with Z-DNA as an example of the latter. Relative interstrand binding energies for six DNA duplex models, two each of configuration I-R (standard WC canonical B-DNA), II-R, and II-L for the duplex d(CGCGAATTCGCG), have been estimated under identical conditions using MM-PBSA analysis from molecular dynamics trajectories using three different AMBER force fields. These simulations support the stereo chemical soundness of configuration II dsDNA forms. Recent force fields (Barcelona Supercomputing Center [BSC] [bsc1] and Olomouc 2015 [OL15]) successfully render stable II-L structures, whereas the previous force field, bsc0, generated stable II-R structures, although with an energy difference between II-R and II-L of ～30?kcal/mol.

Communicated by Ramaswamy H. Sarma     

Flexibility of the Bacterial Chaperone Trigger Factor in Microsecond-Timescale Molecular Dynamics Simulations

The bacterial chaperone trigger factor (TF) is the first chaperone to be encountered by a nascent protein chain as it emerges from the ribosome exit tunnel. Experimental results suggest that TF possesses considerable conformational flexibility, and in an attempt to provide an atomic-level view of this flexibility, we have performed independent 1.5-μs molecular dynamics simulations of TF in explicit solvent using two different simulation force fields (OPLS-AA/L and AMBER ff99SB-ILDN). Both simulations indicate that TF possesses tremendous flexibility, with huge excursions from the crystallographic conformation caused by reorientations of the protein’s constituent domains; both simulations also predict the formation of extensive contacts between TF’s PPIase domain and the Arm 1 domain that is involved in nascent-chain binding. In the OPLS simulation, however, TF rapidly settles into a very compact conformation that persists for at least 1 μs, whereas in the AMBER simulation, it remains highly dynamic; additional simulations in which the two force fields were swapped suggest that these differences are at least partly attributable to sampling issues. The simulation results provide potential rationalizations of a number of experimental observations regarding TF’s conformational behavior and have implications for using simulations to model TF’s function on translating ribosomes.     

Refinement of the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers

We present here the parmbsc0 force field, a refinement of the AMBER parm99 force field, where emphasis has been made on the correct representation of the alpha/gamma concerted rotation in nucleic acids (NAs). The modified force field corrects overpopulations of the alpha/gamma = (g+,t) backbone that were seen in long (more than 10 ns) simulations with previous AMBER parameter sets (parm94-99). The force field has been derived by fitting to high-level quantum mechanical data and verified by comparison with very high-level quantum mechanical calculations and by a very extensive comparison between simulations and experimental data. The set of validation simulations includes two of the longest trajectories published to date for the DNA duplex (200 ns each) and the largest variety of NA structures studied to date (15 different NA families and 97 individual structures). The total simulation time used to validate the force field includes near 1 mus of state-of-the-art molecular dynamics simulations in aqueous solution.