Temperature Replica-exchange Molecular Dynamics Simulations Research Articles

Structural Space of the Duffy Antigen/Receptor for Chemokines' Intrinsically Disordered Ectodomain 1 Explored by Temperature Replica-Exchange Molecular Dynamics Simulations.

Plasmodium vivax malaria affects 14 million people each year. Its invasion requires interactions between the parasitic Duffy-binding protein (PvDBP) and the N-terminal extracellular domain (ECD1) of the host's Duffy antigen/receptor for chemokines (DARC). ECD1 is highly flexible and intrinsically disordered, therefore it can adopt different conformations. We computationally modeled the challenging ECD1 local structure. With T-REMD simulations, we sampled its dynamic behavior and collected its most representative conformations. Our results suggest that most of the DARC ECD1 domain remains in a disordered state during the simulated time. Globular local conformations are found in the analyzed local free-energy minima. These globular conformations share an α-helix spanning residues Ser18 to Ser29 and in many cases they comprise an antiparallel β-sheet, whose β-strands are formed around residues Leu10 and Ala49. The formation of a parallel β-sheet is almost negligible. So far, progress in understanding the mechanisms forming the basis of the P. vivax malaria infection of reticulocytes has been hampered by experimental difficulties, along with a lack of DARC structural information. Our collection of the most probable ECD1 structural conformations will help to advance modeling of the DARC structure and to explore DARC-ECD1 interactions with a range of physiological and pathological ligands.

Conformational dynamics of the cytoplasmic cap during desensitization of the hP2X3R.

The human P2X3 receptor (P2X3R) is a trimeric ligand-gated ion channel activated by extracellular ATP. X-ray crystallography revealed atomic structures of its apo (closed, resting), ATP-bound (open, activated), and ATP-bound (closed, desensitized) states. An intracellular ‘cytoplasmic cap’ was found to stabilize the open pore in the ATP-bound state. However, the cytoplasmic cap was not resolved in the resting or desensitized structures, suggesting a model in which transient formation of the cytoplasmic cap is associated with the opening of the ion pore. To study the functional coupling between (un)folding of the cytoplasmic cap and opening/closing of the P2X3R ion pore, we performed extensive all-atom molecular dynamics (MD) simulations combined with voltage-clamp electrophysiology and mutagenesis. Temperature replica-exchange MD simulations of P2X3R revealed the cytoplasmic cap's unfolding pathway and consequent ion pore closure. Our results show that the N- and C-terminal layers that form the cytoplasmic cap in the open state do not fully unfold during desensitization. By contrast, the partial unfolding of the lower C-terminal layer is sufficient to initiate pore closure/desensitization. In contrast, the N-terminal layers remain folded and keep their spatial proximity to the membrane. Interestingly, not all C-terminal secondary structures unfold, and some structural organization persists, which may allow for rapid transitions between the apo, open, and resting states. The main trigger of pore closure/desensitization is the loss of contact between the C-terminal and the N-terminal part of the cap. We investigated point mutations, aimed at (de)stabilizing the cytoplasmic cap, using free-energy calculations and electrophysiological recordings. These demonstrate that the kinetics of P2X3R desensitization depends on the folding-free energy of the cytoplasmic cap. In summary, our work reveals an allosteric coupling mechanism between the cytoplasmic cap and gating of the transmembrane ion pore in P2X receptors.

Secondary structure dependence on simulation techniques and force field parameters: from disordered to ordered proteins.

Computer simulations are used for identifying the secondary structure properties of ordered and disordered proteins. However, our recent studies showed that the chosen computer simulation protocol, simulation technique, and force field parameter set for a disordered protein impact its predicted secondary structure properties. Here, we compare the outcome from computer simulations utilizing molecular dynamics simulations without parallel tempering techniques using various force field parameter sets and temperature-replica exchange molecular dynamics simulations both for a model ordered and two model disordered proteins. Specifically, the model ordered protein is the third IgG-binding domain of Protein G (GB3) and the two model disordered proteins are amyloid-β(1-40) and α-synuclein in water. Our findings clearly indicate that temperature-replica exchange molecular dynamics simulations and molecular dynamics simulations without special sampling techniques yield similar results for the ordered GB3 protein whereas such agreement between simulation techniques using various force field parameter sets could not be obtained for disordered proteins. These findings clearly indicate that a consensus has to be reached via further development in computer simulation technique and force field parameter sets for disordered proteins.

Deciphering nucleotide modification-induced structure and stability changes

ABSTRACT Nucleotide modification in RNA controls a bevy of biological processes, including RNA degradation, gene expression, and gene editing. In turn, misregulation of modified nucleotides is associated with a host of chronic diseases and disorders. However, the molecular mechanisms driving these processes remain poorly understood. To partially address this knowledge gap, we used alchemical and temperature replica exchange molecular dynamics (TREMD) simulations on an RNA duplex and an analogous hairpin to probe the structural effects of modified and/or mutant nucleotides. The simulations successfully predict the modification/mutation-induced relative free energy change for complementary duplex formation, and structural analyses highlight mechanisms driving stability changes. Furthermore, TREMD simulations for a hairpin-forming RNA with and without modification provide reliable estimations of the energy landscape. Illuminating the impact of methylated and/or mutated nucleotides on the structure-function relationship and the folding energy landscape, the simulations provide insights into modification-induced alterations to the folding mechanics of the hairpin. The results here may be biologically significant as hairpins are widespread structure motifs that play critical roles in gene expression and regulation. Specifically, the tetraloop of the probed hairpin is phylogenetically abundant, and the stem mirrors a miRNA seed region whose modification has been implicated in epilepsy pathogenesis.

Importance of the Force Field Choice in Capturing Functionally Relevant Dynamics in the von Willebrand Factor.

Whether recent updates and new releases of atomistic force fields can model the structural and dynamical properties of proteins containing both folded and partially disordered domains is still unclear. To address this fundamental question, we tested eight recently released force fields against our set of nuclear magnetic resonance (NMR) observables for a complex and medically relevant system, the major factor VIII binding region on the von Willebrand factor. This biomedically important region comprises both a folded and a partially structured domain. By using an enhanced sampling technique (temperature replica-exchange molecular dynamics simulations), we find that some force fields indeed rise to the challenge and capture the structural and dynamical features of the NMR ensemble and, therefore, are the appropriate choice for simulations of proteins with partially structured domains. What is more, we show that only such force fields can qualitatively capture the effects of a pathogenic mutation on the structural ensemble.

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides.

A synthetic riboswitch N1, inserted into the 5′-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6′ hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6′-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

Preferred conformations ofN-glycan core pentasaccharide in solution and in glycoproteins

N-linked glycans are on protein surfaces and have direct and water/ion-mediated interactions with surrounding amino acids. Such contacts could restrict their conformational freedom compared to the same glycans free in solution. In this work, we have examined the conformational freedom of the N-glycan core pentasaccharide moiety in solution using standard molecular dynamics (MD) simulations as well as temperature replica-exchange MD simulations. Both simulations yield the comparable conformational variability of the pentasaccharide in solution, indicating the convergence of both simulations. The glycoprotein crystal structures are analyzed to compare the conformational freedom of the N-glycan on the protein surface with the simulation result. Surprisingly, the pentasaccharide free in solution shows more restricted conformational variability than the N-glycan on the protein surface. The interactions between the carbohydrate and the protein side chain appear to be responsible for the increased conformational diversity of the N-glycan on the protein surface. Finally, the transfer entropy analysis of the simulation trajectory also reveals an unexpected causality relationship between intramolecular hydrogen bonds and the conformational states in that the hydrogen bonds play a role in maintaining the conformational states rather than driving the change in glycosidic torsional states.

De Novo Folding of RNA Hairpins by Temperature Replica Exchange

Describing folding and thermodynamic behaviors of small RNA hairpins is a key step in elucidating the folding processes of larger RNA macromolecules. Experimental methods have been used to describe a wide range of dynamical behavior in RNA hairpins including the dynamics of loop residue interactions and folding rates of stems, but many questions surrounding the detailed mechanism of RNA hairpin formation remain unanswered. The order and directionality of stem nucleation and stem base-pair formation remains open for debate, as well as the effects of stem-length on the rate of hairpin formation and the frequency of base mismatches. All-atom molecular dynamics offers a methodology for analyzing heterogeneous, multiple-pathway folding processes and how they contribute to the overall thermodynamic behaviors of RNA hairpins. To that end, we analyzed the equilibrium folding of a hyperstable tetraloop with a four base-pair stem, r(ggccGCAAggcc), using recently published RNA parameters. Using microsecond-timescale temperature replica-exchange molecular dynamics simulations we are able to sample a large ensemble of conformational states adopted by RNA hairpins and describe the thermodynamic processes involved in hairpin formation. The results give us insights into the biophysical driving forces of RNA folding.

How Kinetics within the Unfolded State Affects Protein Folding: An Analysis Based on Markov State Models and an Ultra-Long MD Trajectory

Understanding how kinetics in the unfolded state affects protein folding is a fundamentally important yet less well-understood issue. Here we employ three different models to analyze the unfolded landscape and folding kinetics of the miniprotein Trp-cage. The first is a 208 μs explicit solvent molecular dynamics (MD) simulation from D. E. Shaw Research containing tens of folding events. The second is a Markov state model (MSM-MD) constructed from the same ultralong MD simulation; MSM-MD can be used to generate thousands of folding events. The third is a Markov state model built from temperature replica exchange MD simulations in implicit solvent (MSM-REMD). All the models exhibit multiple folding pathways, and there is a good correspondence between the folding pathways from direct MD and those computed from the MSMs. The unfolded populations interconvert rapidly between extended and collapsed conformations on time scales ≤40 ns, compared with the folding time of ∼5 μs. The folding rates are independent of where the folding is initiated from within the unfolded ensemble. About 90% of the unfolded states are sampled within the first 40 μs of the ultralong MD trajectory, which on average explores ∼27% of the unfolded state ensemble between consecutive folding events. We clustered the folding pathways according to structural similarity into "tubes", and kinetically partitioned the unfolded state into populations that fold along different tubes. From our analysis of the simulations and a simple kinetic model, we find that, when the mixing within the unfolded state is comparable to or faster than folding, the folding waiting times for all the folding tubes are similar and the folding kinetics is essentially single exponential despite the presence of heterogeneous folding paths with nonuniform barriers. When the mixing is much slower than folding, different unfolded populations fold independently, leading to nonexponential kinetics. A kinetic partition of the Trp-cage unfolded state is constructed which reveals that different unfolded populations have almost the same probability to fold along any of the multiple folding paths. We are investigating whether the results for the kinetics in the unfolded state of the 20-residue Trp-cage is representative of larger single domain proteins.

Probing the free energy landscape of the FBP28WW domain using multiple techniques

The free-energy landscape of a small protein, the FBP 28 WW domain, has been explored using molecular dynamics (MD) simulations with alternative descriptions of the molecule. The molecular models used range from coarse-grained to all-atom with either an implicit or explicit treatment of the solvent. Sampling of conformation space was performed using both conventional and temperature-replica exchange MD simulations. Experimental chemical shifts and NOEs were used to validate the simulations, and experimental phi values both for validation and as restraints. This combination of different approaches has provided insight into the free energy landscape and barriers encountered by the protein during folding and enabled the characterization of native, denatured and transition states which are compatible with the available experimental data. All the molecular models used stabilize well defined native and denatured basins; however, the degree of agreement with the available experimental data varies. While the most detailed, explicit solvent model predicts the data reasonably accurately, it does not fold despite a simulation time 10 times that of the experimental folding time. The less detailed models performed poorly relative to the explicit solvent model: an implicit solvent model stabilizes a ground state which differs from the experimental native state, and a structure-based model underestimates the size of the barrier between the two states. The use of experimental phi values both as restraints, and to extract structures from unfolding simulations, result in conformations which, although not necessarily true transition states, appear to share the geometrical characteristics of transition state structures. In addition to characterizing the native, transition and denatured states of this particular system in this work, the advantages and limitations of using varying levels of representation are discussed.

A temperature predictor for parallel tempering simulations

An algorithm is proposed that generates a set of temperatures for use in parallel tempering simulations (also known as temperature-replica exchange molecular dynamics simulations) of proteins to obtain a desired exchange probability Pdes. The input consists of the number of protein atoms and water molecules in the system, information about the use of constraints and virtual sites and the lower temperature limits. The temperatures generated yield probabilities which are very close to Pdes (correlation 97%), independent of force field and over a wide temperature range. To facilitate its use, the algorithm has been implemented as a web server at .

Convergence and sampling efficiency in replica exchange simulations of peptide folding in explicit solvent

Replica exchange methods (REMs) are increasingly used to improve sampling in molecular dynamics (MD) simulations of biomolecular systems. However, despite having been shown to be very effective on model systems, the application of REM in complex systems such as for the simulation of protein and peptide folding in explicit solvent has not been objectively tested in detail. Here we present a comparison of conventional MD and temperature replica exchange MD (T-REMD) simulations of a beta-heptapeptide in explicit solvent. This system has previously been shown to undergo reversible folding on the time scales accessible to MD simulation and thus allows a direct one-to-one comparison of efficiency. The primary properties compared are the free energy of folding and the relative populations of different conformers as a function of temperature. It is found that to achieve a similar degree of precision T-REMD simulations starting from a random set of initial configurations were approximately an order of magnitude more computationally efficient than a single 800 ns conventional MD simulation for this system at the lowest temperature investigated (275 K). However, whereas it was found that T-REMD simulations are more than four times more efficient than multiple independent MD simulations at one temperature (300 K) the actual increase in conformation sampling was only twofold. The overall gain in efficiency using REMD resulted primarily from the ordering of different conformational states over temperature, as opposed to a large increase of conformational sampling. It is also shown that in this system exchanges are accepted primarily based on (random) fluctuations within the solvent and are not strongly correlated with the instantaneous peptide conformation raising questions in regard to the efficiency of T-REMD in larger systems.