Lillian T. Chong

Simulations of the Alternating Access Mechanism of the Sodium Symporter Mhp1

Sodium coupled cotransporters of the five-helix inverted repeat (5HIR) superfamily use an alternating access mechanism to transport a myriad of small molecules across the cell membrane. One of the primary steps in this mechanism is the conformational transition from a state poised to bind extracellular substrates to a state that is competent to deliver substrate to the cytoplasm. Here, we construct a coarse-grained model of the 5HIR benzylhydantoin transporter Mhp1 that incorporates experimental structures of the outward- and inward-open states to investigate the mechanism of this conformational change. Using the weighted ensemble path-sampling method, we rigorously sample the outward- to inward-facing transition path ensemble. The transition path ensemble reveals a heterogeneous set of pathways connecting the two states and identifies two modes of transport: one consistent with a strict alternating access mechanism and another where decoupling of the inner and outer gates causes the transient formation of a continuous permeation pathway through the transporter. We also show that the conformational switch between the outward- and inward-open states results from rigid body motions of the hash motif relative to the substrate bundle, supporting the rocking bundle hypothesis. Finally, our methodology provides the groundwork for more chemically detailed investigations of the alternating mechanism.

Effect of interdomain linker length on an antagonistic folding-unfolding equilibrium between two protein domains.

Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (DeltaG(X)). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. DeltaG(X) values are determined by measuring the extent to which Co(2+) binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that DeltaG(X) increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.

Evaluating the Strength of Salt Bridges: A Comparison of Current Biomolecular Force Fields

Recent advances in computer hardware and software have made rigorous evaluation of current biomolecular force fields using microsecond-scale simulations possible. Force fields differ in their treatment of electrostatic interactions, including the formation of salt bridges in proteins. Here we conducted an extensive evaluation of salt bridge interactions in the latest AMBER, CHARMM, and OPLS force fields, using microsecond-scale molecular dynamics simulations of amino acid analogues in explicit solvent. We focused on salt bridges between three different pairs of oppositely charged amino acids: Arg/Asp, Lys/Asp, and His(+)/Asp. Our results reveal considerable variability in the predicted KA values of the salt bridges for these force fields, as well as differences from experimental data: almost all of the force fields overestimate the strengths of the salt bridges. When amino acids are represented by side-chain analogues, the AMBER ff03 force field overestimates the KA values the least, while for complete amino acids, the AMBER ff13α force field yields the lowest KA value, most likely caused by an altered balance of side-chain/side-chain and side-chain/backbone contacts. These findings confirm the notion that the implicit incorporation of solvent polarization improves the accuracy of modeling salt bridge interactions.

WESTPA: An Interoperable, Highly Scalable Software Package for Weighted Ensemble Simulation and Analysis

The weighted ensemble (WE) path sampling approach orchestrates an ensemble of parallel calculations with intermittent communication to enhance the sampling of rare events, such as molecular associations or conformational changes in proteins or peptides. Trajectories are replicated and pruned in a way that focuses computational effort on underexplored regions of configuration space while maintaining rigorous kinetics. To enable the simulation of rare events at any scale (e.g., atomistic, cellular), we have developed an open-source, interoperable, and highly scalable software package for the execution and analysis of WE simulations: WESTPA (The Weighted Ensemble Simulation Toolkit with Parallelization and Analysis). WESTPA scales to thousands of CPU cores and includes a suite of analysis tools that have been implemented in a massively parallel fashion. The software has been designed to interface conveniently with any dynamics engine and has already been used with a variety of molecular dynamics (e.g., GROMACS, NAMD, OpenMM, AMBER) and cell-modeling packages (e.g., BioNetGen, MCell). WESTPA has been in production use for over a year, and its utility has been demonstrated for a broad set of problems, ranging from atomically detailed host–guest associations to nonspatial chemical kinetics of cellular signaling networks. The following describes the design and features of WESTPA, including the facilities it provides for running WE simulations and storing and analyzing WE simulation data, as well as examples of input and output.

Simultaneous Computation of Dynamical and Equilibrium Information Using a Weighted Ensemble of Trajectories.

Equilibrium formally can be represented as an ensemble of uncoupled systems undergoing unbiased dynamics in which detailed balance is maintained. Many nonequilibrium processes can be described by suitable subsets of the equilibrium ensemble. Here, we employ the “weighted ensemble” (WE) simulation protocol [Huber and Kim, Biophys. J.1996, 70, 97–110] to generate equilibrium trajectory ensembles and extract nonequilibrium subsets for computing kinetic quantities. States do not need to be chosen in advance. The procedure formally allows estimation of kinetic rates between arbitrary states chosen after the simulation, along with their equilibrium populations. We also describe a related history-dependent matrix procedure for estimating equilibrium and nonequilibrium observables when phase space has been divided into arbitrary non-Markovian regions, whether in WE or ordinary simulation. In this proof-of-principle study, these methods are successfully applied and validated on two molecular systems: explicitly solvated methane association and the implicitly solvated Ala4 peptide. We comment on challenges remaining in WE calculations.

Efficient Explicit-Solvent Molecular Dynamics Simulations of Molecular Association Kinetics: Methane/Methane, Na+/Cl−, Methane/Benzene, and K+/18-Crown-6 Ether

Atomically detailed views of molecular recognition events are of great interest to a variety of research areas in biology and chemistry. Here, we apply the weighted ensemble path sampling approach to improve the efficiency of explicit solvent molecular dynamics (MD) simulations in sampling molecular association events between two methane molecules, Na(+) and Cl(-) ions, methane and benzene, and the K(+) ion and 18-crown-6 ether. Relative to brute force simulation, we obtain efficiency gains of at least 300 and 1100-fold for the most challenging system, K(+)/18-crown-6 ether, in terms of sampling the association rate constant k and distribution of times required to traverse transition paths, respectively. Our results indicate that weighted ensemble sampling is likely to allow for even greater efficiencies for more complex systems with higher barriers to molecular association.

The Native GCN4 Leucine-Zipper Domain Does Not Uniquely Specify a Dimeric Oligomerization State

The dimerization domain of the yeast transcription factor GCN4, one of the first coiled-coil proteins to be structurally characterized at high resolution, has served as the basis for numerous fundamental studies on α-helical folding. Mutations in the GCN4 leucine zipper are known to change its preferred oligomerization state from dimeric to trimeric or tetrameric; however, the wild-type sequence has been assumed to encode a two-chain assembly exclusively. Here we demonstrate that the GCN4 coiled-coil domain can populate either a dimer or trimer fold, depending on environment. We report high-resolution crystal structures of the wild-type sequence in dimeric and trimeric assemblies. Biophysical measurements suggest populations of both oligomerization states under certain experimental conditions in solution. We use parallel tempering molecular dynamics simulations on the microsecond time scale to compare the stability of the dimer and trimer folded states in isolation. In total, our results suggest that the folding behavior of the well-studied GCN4 leucine-zipper domain is more complex than was previously appreciated. Our results have implications in ongoing efforts to establish predictive algorithms for coiled-coil folds and the selection of coiled-coil model systems for design and mutational studies where oligomerization state specificity is an important consideration.

Further along the Road Less Traveled: AMBER ff15ipq, an Original Protein Force Field Built on a Self-Consistent Physical Model.

We present the AMBER ff15ipq force field for proteins, the second-generation force field developed using the Implicitly Polarized Q (IPolQ) scheme for deriving implicitly polarized atomic charges in the presence of explicit solvent. The ff15ipq force field is a complete rederivation including more than 300 unique atomic charges, 900 unique torsion terms, 60 new angle parameters, and new atomic radii for polar hydrogens. The atomic charges were derived in the context of the SPC/Eb water model, which yields more-accurate rotational diffusion of proteins and enables direct calculation of nuclear magnetic resonance (NMR) relaxation parameters from molecular dynamics simulations. The atomic radii improve the accuracy of modeling salt bridge interactions relative to contemporary fixed-charge force fields, rectifying a limitation of ff14ipq that resulted from its use of pair-specific Lennard-Jones radii. In addition, ff15ipq reproduces penta-alanine J-coupling constants exceptionally well, gives reasonable agreement with NMR relaxation rates, and maintains the expected conformational propensities of structured proteins/peptides, as well as disordered peptides—all on the microsecond (μs) time scale, which is a critical regime for drug design applications. These encouraging results demonstrate the power and robustness of our automated methods for deriving new force fields. All parameters described here and the mdgx program used to fit them are included in the AmberTools16 distribution.

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations.

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.

Effects of high temperature on desolvation costs of salt bridges across protein binding interfaces: similarities and differences between implicit and explicit solvent models.

The role of salt bridges in protein-protein binding is largely determined by the costs of desolvating the oppositely charged members of the salt bridge upon binding. On the basis of Poisson-Boltzmann (PB) implicit solvent calculations, it has been proposed that the reduced desolvation penalties of salt bridges at high temperatures provide one explanation for the increased abundance of salt bridges in hyperthermophilic proteins. Here, for the first time, we directly compare the PB implicit solvent model with several explicit water models in computing the effects of extremely high temperature (i.e., 100 °C) on the desolvation penalties of salt bridges across protein-protein interfaces. With the exception of two outliers, the desolvation costs at 100 °C from implicit and explicit solvent calculations are of similar magnitudes and significantly reduced relative to 25 °C. The two outliers correspond to salt bridges that are both buried and part of a salt bridge network, a challenging case that should be considered in the development of fast solvation models.