Ab initio RNA folding by discrete molecular dynamics: From structure prediction to folding mechanisms

We have applied all atoms discrete molecular dynamics (DMD) based on a quasi-physical potential to study the flexibility of an extended set of proteins for which atomistic MD simulations are available. The method uses pure physical potentials supplemented by information on secondary structure and despite its simplicity is able to reproduce with good accuracy the dynamics of proteins in solution. The method presents a clear improvement with respect to coarse-grained methods based on structure potentials and opens the possibility to explore dynamics of proteins out from the equilibrium and to trace conformational changes induced by interaction of proteins with both small and macromolecular ligands.

α-Synuclein (α-syn) is the major component of the intraneuronal inclusions called Lewy bodies, which are the pathological hallmark of Parkinson's disease. α-Syn is capable of self-assembly into many different species, such as soluble oligomers and fibrils. Even though attempts to resolve the structures of the protein have been made, detailed understanding about the structures and their relationship with the different aggregation steps is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson's disease. Here we report the structural flexibility of α-syn monomers and dimers in an aqueous solution environment as probed by single-molecule time-lapse high-speed AFM. In addition, we present the molecular basis for the structural transitions using discrete molecular dynamics (DMD) simulations. α-Syn monomers assume a globular conformation, which is capable of forming tail-like protrusions over dozens of seconds. Importantly, a globular monomer can adopt fully extended conformations. Dimers, on the other hand, are less dynamic and show a dumbbell conformation that experiences morphological changes over time. DMD simulations revealed that the α-syn monomer consists of several tightly packed small helices. The tail-like protrusions are also helical with a small β-sheet, acting as a "hinge". Monomers within dimers have a large interfacial interaction area and are stabilized by interactions in the non-amyloid central (NAC) regions. Furthermore, the dimer NAC-region of each α-syn monomer forms a β-rich segment. Moreover, NAC-regions are located in the hydrophobic core of the dimer.

The abnormal aggregation of human calcitonin (hCT) hormone peptides impairs their physiological function, leading to harmful immune responses and cytotoxicity, which limits their clinical utility. Interestingly, a representative hCT analog incorporating Y12L and N17H substitutions (DM-hCT) has shown reduced aggregation tendencies while maintaining bioactivity. But the molecular mechanism of Y12L and N17H substitutions on the conformational dynamics of hCT remains unclear. Here, we systematically investigated the folding and self-assembly dynamics of hCT and DM-hCT using atomistic discrete molecular dynamics (DMD) simulations. Our findings revealed that hCT monomers predominantly adopted unstructured conformations with dynamic helices. Oligomerization of hCT resulted in the formation of β-sheet-rich aggregates and β-barrel intermediates. The Y12L and N17H substitutions enhanced helical conformations and suppressed β-sheet formation in both monomers and oligomers. These substitutions stabilized the dynamic helices and disrupted aromatic interactions responsible for β-sheet formation at residue 12. Notably, DM-hCT assemblies still exhibited β-sheets in phenylalanine-rich and C-terminal hydrophobic regions, suggesting that future optimizations should focus on these areas. Our simulations provide insights into the molecular mechanisms underlying hCT aggregation and the amyloid-resistant effects of Y12L and N17H substitutions. These findings have valuable implications for the development of clinical hCT analogs.

Computational insights into the aggregation mechanism of human calcitonin.

Experimental findings suggest that oligomeric forms of the amyloid beta protein (Abeta) play a critical role in Alzheimer's disease. Thus, elucidating their structure and the mechanisms of their formation is critical for developing therapeutic agents. We use discrete molecular dynamics simulations and a four-bead protein model to study oligomerization of two predominant alloforms, Abeta40 and Abeta42, at the atomic level. The four-bead model incorporates backbone hydrogen-bond interactions and amino acid-specific interactions mediated through hydrophobic and hydrophilic elements of the side chains. During the simulations we observe monomer folding and aggregation of monomers into oligomers of variable sizes. Abeta40 forms significantly more dimers than Abeta42, whereas pentamers are significantly more abundant in Abeta42 relative to Abeta40. Structure analysis reveals a turn centered at Gly-37-Gly-38 that is present in a folded Abeta42 monomer but not in a folded Abeta40 monomer and is associated with the first contacts that form during monomer folding. Our results suggest that this turn plays an important role in Abeta42 pentamer formation. Abeta pentamers have a globular structure comprising hydrophobic residues within the pentamer's core and hydrophilic N-terminal residues at the surface of the pentamer. The N termini of Abeta40 pentamers are more spatially restricted than Abeta42 pentamers. Abeta40 pentamers form a beta-strand structure involving Ala-2-Phe-4, which is absent in Abeta42 pentamers. These structural differences imply a different degree of hydrophobic core exposure between pentamers of the two alloforms, with the hydrophobic core of the Abeta42 pentamer being more exposed and thus more prone to form larger oligomers.

The RNA folding trajectory features numerous off-pathway folding traps, which represent conformations that are often equally as stable as the native functional ones. Therefore, the conversion between these off-pathway structures and the native correctly folded ones is the critical step in RNA folding. This process, referred to as RNA refolding, is slow, and is represented by a transition state that has a characteristic high free energy. Because this kinetically limiting process occurs in vivo, proteins (called RNA chaperones) have evolved that facilitate the (re)folding of RNA molecules. Here, we present an overview of how proteins interact with RNA molecules in order to achieve properly folded states. In this respect, the discrimination between static and transient interactions is crucial, as different proteins have evolved a multitude of mechanisms for RNA remodeling. For RNA chaperones that act in a sequence-unspecific manner and without the use of external sources of energy, such as ATP, transient RNA–protein interactions represent the basis of the mode of action. By presenting stretches of positively charged amino acids that are positioned in defined spatial configurations, RNA chaperones enable the RNA backbone, via transient electrostatic interactions, to sample a wider conformational space that opens the route for efficient refolding reactions.

RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop-receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21-47 degrees C), from which a van't Hoff analysis yields the enthalpy (DeltaH degrees) and entropy (DeltaS degrees) of docking. Tetraloop-receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl(2) and 100 mM NaCl, with excellent agreement between immobilized (DeltaH degrees = -17.4 +/- 1.6 kcal/mol, and DeltaS degrees = -56.2 +/- 5.4 cal mol(-1) K(-1)) and freely diffusing (DeltaH degrees = -17.2 +/- 1.6 kcal/mol, and DeltaS degrees = -55.9 +/- 5.2 cal mol(-1) K(-1)) species. Kinetic heterogeneity in the tetraloop-receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop-receptor interaction can account for approximately 60% of the DeltaH degrees and DeltaS degrees of P4-P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop-receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions.

The mechanical behavior of nanoparticle assemblies depends strongly on particle size, yet the underlying mechanisms remain insufficiently understood. In present study, we employ a scheme combining discrete element method (DEM) and molecular dynamics (MD) simulations to examine size-dependent strength and deformation in disordered copper nanoparticle assemblies. Granular packings generated by DEM were transformed into atomic models and subjected to uniaxial compression in MD simulations. Assemblies composed of nanoparticles with radius smaller than ~2.5 nm fully densify during relaxation, forming nanopolycrystalline solids, whereas larger particles preserve porous architectures. This structural divergence governs subsequent deformation. Small-particle assemblies deform through grain boundary migration and grain growth, exhibiting an inverse Hall–Petch-type strength dependence. In contrast, large-particle assemblies deform primarily via interparticle contact evolution and densification, with strength conforming to a Gibson–Ashby-type prediction. A scaling law captures the strength variation across size range in this regime. These results establish the competition between surface energy-driven densification and contact-dominated deformation as the controlling factor in the mechanical response of nanoparticle assemblies, providing guidance for designing nanoparticle-based materials with tailored mechanical performance.

A transition in hydraulically induced granular displacement patterns is studied by means of discrete numerical molecular dynamics simulations. During this transition the patterns change from fractures and fingers to finely dispersed bubbles. The dynamics of the displacement patterns are studied in a rectangular Hele-Shaw cell filled with a dense but permeable two-dimensional granular layer. At one side of the cell the pressure of the compressible interstitial gas is increased. At the opposite side from the inlet of the cell a semipermeable boundary is located. This boundary is only permeable towards the gas phase while preventing grains from leaving the cell. The imposed pressure gradient compacts the grains. In the process we can identify and describe a mechanism that controls the transition of the emerging displacement patterns from fractures and fingers to finely dispersed bubbles as a function of the interstitial gas's properties and the characteristics of the granular phase.

Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177–182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure.

How ions affect RNA folding thermodynamics and kinetics is an important but a vexing problem that remains unsolved. Experiments have shown that the free-energy change, Δ G( c), of RNA upon folding varies with the salt concentration ( c) as, Δ G( c) = k c ln c + const, where the coefficient k c is proportional to the difference in the ion preferential coefficient, ΔΓ. We performed simulations of a coarse-grained model, by modeling electrostatic interactions implicitly and with explicit representation of ions, to elucidate the molecular underpinnings of the relationship between Δ G and ΔΓ. The simulations quantitatively reproduce the heat capacity for a pseudoknot, thus validating the model. We show that Δ G( c), calculated directly from ΔΓ, varies linearly with ln c ( c < 0.2 M), for a hairpin and the pseudoknot, demonstrating a molecular link between the two quantities. Explicit ion simulations also show the linear dependence of Δ G( c) on ln c at all c with k c = 2 kB T, except that Δ G( c) values are shifted by ∼2 kcal/mol higher than experiments. The discrepancy is due to an underestimation of Γ for both the folded and unfolded states while giving accurate values for ΔΓ. The predictions for the salt dependence of ΔΓ are amenable to test using single-molecule pulling experiments. The framework provided here can be used to obtain accurate thermodynamics for other RNA molecules as well.

Multiscale Modeling of Nucleosome Dynamics

Transitions between different conformational states, so-called conformational switching, are intrinsic to RNA catalytic and regulatory functions. Often, conformational switching occurs on time scales of several seconds. In combination with the recent real-time NMR experiments (Wenter et al. Angew. Chem. Int. Ed. 2005, 44, 2600; Wenter et al. ChemBioChem 2006, 7, 417) for the transitions between bistable RNA conformations, we combine the master equation method with the kinetic cluster method to investigate the detailed kinetic mechanism and the factors that govern the folding kinetics. We propose that heat capacity change (ΔC(p)) upon RNA folding may be important for RNA folding kinetics. In addition, we find that, for tetraloop hairpins, noncanonical (tertiary) intraloop interactions are important to determine the folding kinetics. Furthermore, through theory-experiment comparisons, we find that the different rate models for the fundamental steps (i.e., formation/disruption of a base pair or stack) can cause contrasting results in the theoretical predictions.

Cellular processes involve large numbers of RNA molecules. The functions of these RNA molecules and their binding to molecular machines are highly dependent on their 3D structures. One of the key challenges in RNA structure prediction and modeling is predicting the spatial arrangement of the various structural elements of RNA. As RNA folding is generally hierarchical, methods involving coarse-grained models hold great promise for this purpose. We present here a novel coarse-grained method for sampling, based on game theory and knowledge-based potentials. This strategy, GARN (Game Algorithm for RNa sampling), is often much faster than previously described techniques and generates large sets of solutions closely resembling the native structure. GARN is thus a suitable starting point for the molecular modeling of large RNAs, particularly those with experimental constraints. GARN is available from: http://garn.lri.fr/.

Medin is a principal component of localized amyloid found in the vasculature of individuals over 50 years old. Its amyloid aggregation has been linked to endothelial dysfunction and vascular inflammation, contributing to the pathogenesis of various vascular diseases. Despite its significance, the structures of the medin monomer, oligomer, and fibril remain elusive, and the dynamic processes of medin aggregation are not fully understood. In this study, we comprehensively investigated the medin folding and dimerization dynamics and conformations using atomistic discrete molecular dynamics simulations. Our simulation results suggested that the folding initiation of the medin involved the formation of β-sheets around medin30-41 and medin42-50, with subsequent capping of other segments to their β-sheet edges. Medin monomers typically consisted of three or four β-strands, along with a dynamic N-terminal helix. Two isolated medin peptides readily aggregated into a β-sheet-rich dimer, displaying a strong aggregation propensity. Dimerization of medin not only enhanced the β-sheet conformations but also led to the formation of β-barrel oligomers. The aggregation tendencies of medin1-18 and medin19-29 were relatively weak. However, the segments of medin30-41 and medin42-50 played a crucial role as they primarily formed a β-sheet core and facilitated medin1-18 and medin19-29 to form intra- and interpeptide β-sheets. The findings highlight the critical role of the medin30-41 and medin42-50 regions in stabilizing the monomer structure and driving the medin amyloid aggregation. These regions could potentially serve as promising targets for designing antiamyloid inhibitors against amyloid aggregation of medin. Additionally, our study provides a full picture of the monomer conformations and dimerization dynamics for medin, which will help better understand the pathology of medin aggregation.