Characterizing the Folding Transition-State Ensembles in the Energy Landscape of an RNA Tetraloop.

Molecular dynamics (MD) simulations have become increasingly powerful and can now describe the folding/unfolding of small biomolecules in atomic detail. However, a major challenge in MD simulations is to represent the complex energy landscape of biomolecules using a small number of reaction coordinates. In this study, we investigate the folding pathways of an RNA tetraloop, gcGCAAgc, using five classical MD simulations with a combined simulation time of approximately 120 µs. Our approach involves analyzing the tetraloop dynamics, including the folding transition state ensembles, using the energy landscape visualization method (ELViM). The ELViM is an approach that uses internal distances to compare any two conformations, allowing for a detailed description of the folding process without requiring root mean square alignment of structures. This method has previously been applied to describe the energy landscape of disordered ß-amyloid peptides and other proteins. The ELViM results in a non-linear projection of the multidimensional space, providing a comprehensive representation of the tetraloop's energy landscape. Our results reveal four distinct transition-state regions and establish the paths that lead to the folded tetraloop structure. This detailed analysis of the tetraloop's folding process has important implications for understanding RNA folding, and the ELViM approach can be used to study other biomolecules.

Millisecond molecular dynamics simulations of KRas-dimer formation and interfaces.

Ras dimers have been proposed as building blocks for initiating the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cellular signaling pathway. To better examine the structure of possible dimer interfaces, the dynamics of Ras dimerization, and its potential signaling consequences, we performed molecular dynamics simulations totaling 1 ms of sampling, using an all-atom model of two full-length, farnesylated, guanosine triphosphate (GTP)-bound, wild-type KRas4b proteins diffusing on 29%POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine)-mixed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membranes. Our simulations unveil an ensemble of thermodynamically weak KRas dimers spanning multiple conformations. The most stable conformations, having the largest interface areas, involve helix α2 and a hypervariable region (HVR). Among the dimer conformations, we found that the HVR of each KRas has frequent interactions with various parts of the dimer, thus potentially mediating the dimerization. Some dimer configurations have one KRas G-domain elevated above the lipid bilayer surface by residing on top of the other G-domain, thus likely contributing to the recruitment of cytosolic Raf kinases in the context of a stably formed multi-protein complex. We identified a variant of the α4-α5 KRas-dimer interface that is similar to the interfaces obtained with fluorescence resonance energy transfer (FRET) data of HRas on lipid bilayers. Interestingly, we found two arginine fingers, R68 and R149, that directly interact with the beta-phosphate of the GTP bound in KRas, in a manner similar to what is observed in a crystal structure of GAP-HRas complex, which can facilitate the GTP hydrolysis via the arginine finger of GTPase-activating protein (GAP).

The Plasma Membrane as a Competitive Inhibitor and Positive Allosteric Modulator of KRas4B Signaling.

Mutant Ras proteins are important drivers of human cancers, yet no approved drugs act directly on this difficult target. Over the last decade, the idea has emerged that oncogenic signaling can be diminished by molecules that drive Ras into orientations in which effector-binding interfaces are occluded by the cell membrane. To support this approach to drug discovery, we characterize the orientational preferences of membrane-bound K-Ras4B in 1.45-ms aggregate time of atomistic molecular dynamics simulations. Individual simulations probe active or inactive states of Ras on membranes with or without anionic lipids. We find that the membrane orientation of Ras is relatively insensitive to its bound guanine nucleotide and activation state but depends strongly on interactions with anionic phosphatidylserine lipids. These lipids slow Ras' translational and orientational diffusion and promote a discrete population in which small changes in orientation control Ras' competence to bind multiple regulator and effector proteins. Our results suggest that compound-directed conversion of constitutively active mutant Ras into functionally inactive forms may be accessible via subtle perturbations of Ras' orientational preferences at the membrane surface.

Presence or Absence of Ras Dimerization Shows Distinct Kinetic Signature in Ras-Raf Interaction.

Initiations of cell signaling pathways often occur through the formation of multiprotein complexes that form through protein-protein interactions. Therefore, detecting their presence is central to understanding the function of a cell signaling pathway, aberration of which often leads to fatal diseases, including cancers. However, the multiprotein complexes are often difficult to detect using microscopes due to their small sizes. Therefore, currently, their presence can be only detected through indirect means. In this article, we propose to investigate the presence or absence of protein complexes through some easily measurable kinetic parameters, such as activation rates. As a proof of concept, we investigate the Ras-Raf system, a well-characterized cell signaling system. It has been hypothesized that Ras dimerization is necessary to create activated Raf dimers. Although there are circumstantial evidences supporting the Ras dimerization hypothesis, direct proof of Ras dimerization is still inconclusive. In the absence of conclusive direct experimental proof, this hypothesis can only be examined through indirect evidences of Ras dimerization. In this article, using a multiscale simulation technique, we provide multiple criteria that distinguishes an activation mechanism involving Ras dimerization from another mechanism that does not involve Ras dimerization. The provided criteria will be useful in the investigation of not only Ras-Raf interaction but also other two-protein interactions.

Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD.

CRAF activation requires binding to membrane-anchored and active GTP-bound RAS. Whereas its RAS-binding domain (RBD) contains the main binding interface to the RAS G domain, its cysteine-rich domain (CRD) is responsible for association to anionic lipid-rich membranes. Both RAF domains are connected by a short linker, and it remains unclear if the two domains act independently or if one domain can impact the function of the other. Here, we used a combination of coarse-grained and all-atom molecular dynamics simulations of a CRAF RBD-CRD construct to investigate the dynamics of the RBD when it is tethered to CRD that is anchored to a POPC:POPS model membrane. First, we show that the RBD positioning is very dynamic with a preferential localization near the membrane surface. Next, we show that membrane-localized RBD has its RAS-binding interface mostly inaccessible because of its proximity to the membrane. Several positively charged residues in this interface were identified from simulations as important for driving RBD association to the membrane. Surface plasmon resonance (SPR) measurements confirmed that mutations of these RBD residues reduced the liposome partitioning of RBD-CRD. Last, simulations indicated that the presence of RBD near the membrane led to a local enrichment of anionic lipids that could potentially enhance the membrane affinity of the entire RBD-CRD construct. This was supported by SPR measurements that showed stronger liposome partitioning of RBD-CRD relative to CRD alone. These findings thus suggest that the RBD and CRD have synergistic effects on their membrane dynamics, with CRD bringing RBD closer to the membrane that impacts its accessibility to RAS and with RBD causing local anionic lipid enrichment that enhances the overall affinity between the membrane and RBD-CRD. These mechanisms have potential implications on the order of events of the interactions between RAS and CRAF at the membrane.

Free-energy landscape of a hyperstable RNA tetraloop.

We report the characterization of the energy landscape and the folding/unfolding thermodynamics of a hyperstable RNA tetraloop obtained through high-performance molecular dynamics simulations at microsecond timescales. Sampling of the configurational landscape is conducted using temperature replica exchange molecular dynamics over three isochores at high, ambient, and negative pressures to determine the thermodynamic stability and the free-energy landscape of the tetraloop. The simulations reveal reversible folding/unfolding transitions of the tetraloop into the canonical A-RNA conformation and the presence of two alternative configurations, including a left-handed Z-RNA conformation and a compact purine Triplet. Increasing hydrostatic pressure shows a stabilizing effect on the A-RNA conformation and a destabilization of the left-handed Z-RNA. Our results provide a comprehensive description of the folded free-energy landscape of a hyperstable RNA tetraloop and highlight the significant advances of all-atom molecular dynamics in describing the unbiased folding of a simple RNA secondary structure motif.

Water Determines the Structure and Dynamics of Proteins.

Water is an essential participant in the stability, structure, dynamics, and function of proteins and other biomolecules. Thermodynamically, changes in the aqueous environment affect the stability of biomolecules. Structurally, water participates chemically in the catalytic function of proteins and nucleic acids and physically in the collapse of the protein chain during folding through hydrophobic collapse and mediates binding through the hydrogen bond in complex formation. Water is a partner that slaves the dynamics of proteins, and water interaction with proteins affect their dynamics. Here we provide a review of the experimental and computational advances over the past decade in understanding the role of water in the dynamics, structure, and function of proteins. We focus on the combination of X-ray and neutron crystallography, NMR, terahertz spectroscopy, mass spectroscopy, thermodynamics, and computer simulations to reveal how water assist proteins in their function. The recent advances in computer simulations and the enhanced sensitivity of experimental tools promise major advances in the understanding of protein dynamics, and water surely will be a protagonist.

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol-1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol-1) than for L67 (58 ± 3 mL mol-1) inteins. The measured difference in ΔVf (13 ± 3 mL mol-1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1H-15N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations.

We report the de novo folding of three hyperstable RNA tetraloops to 1-3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra- and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations.

Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor?

G-protein-coupled receptors are eukaryotic membrane proteins with broad biological and pharmacological relevance. Like all membrane-embedded proteins, their location and orientation are influenced by lipids, which can also impact protein function via specific interactions. Extensive simulations totaling 0.25 ms reveal a process in which phospholipids from the membrane's cytosolic leaflet enter the empty G-protein binding site of an activated ß2 adrenergic receptor and form salt-bridge interactions that inhibit ionic lock formation and prolong active-state residency. Simulations of the receptor embedded in an anionic membrane show increased lipid binding, providing a molecular mechanism for the experimental observation that anionic lipids can enhance receptor activity. Conservation of the arginine component of the ionic lock among Rhodopsin-like G-protein-coupled receptors suggests that intracellular lipid ingression between receptor helices H6 and H7 may be a general mechanism for active-state stabilization.

HIV-1 Tat membrane interactions probed using X-ray and neutron scattering, CD spectroscopy and MD simulations.

We report the effect on lipid bilayers of the Tat peptide Y47GRKKRRQRRR57 from the HIV-1 virus transactivator of translation (Tat) protein. Synergistic use of low-angle X-ray scattering (LAXS) and atomistic molecular dynamic simulations (MD) indicate Tat peptide binding to neutral dioleoylphosphocholine (DOPC) lipid headgroups. This binding induced the local lipid phosphate groups to move 3Å closer to the center of the bilayer. Many of the positively charged guanidinium components of the arginines were as close to the center of the bilayer as the locally thinned lipid phosphate groups. LAXS data for DOPC, DOPC/dioleoylphosphoethanolamine (DOPE), DOPC/dioleoylphosphoserine (DOPS), and a mimic of the nuclear membrane gave similar results. Generally, the Tat peptide decreased the bilayer bending modulus KC and increased the area/lipid. Further indications that Tat softens a membrane, thereby facilitating translocation, were provided by wide-angle X-ray scattering (WAXS) and neutron scattering. CD spectroscopy was also applied to further characterize Tat/membrane interactions. Although a mechanism for translation remains obscure, this study suggests that the peptide/lipid interaction makes the Tat peptide poised to translocate from the headgroup region.

Efficient Schmidt number scaling in dissipative particle dynamics.

Dissipative particle dynamics is a widely used mesoscale technique for the simulation of hydrodynamics (as well as immersed particles) utilizing coarse-grained molecular dynamics. While the method is capable of describing any fluid, the typical choice of the friction coefficient γ and dissipative force cutoff rc yields an unacceptably low Schmidt number Sc for the simulation of liquid water at standard temperature and pressure. There are a variety of ways to raise Sc, such as increasing γ and rc, but the relative cost of modifying each parameter (and the concomitant impact on numerical accuracy) has heretofore remained undetermined. We perform a detailed search over the parameter space, identifying the optimal strategy for the efficient and accuracy-preserving scaling of Sc, using both numerical simulations and theoretical predictions. The composite results recommend a parameter choice that leads to a speed improvement of a factor of three versus previously utilized strategies.

Mechanism of enhanced mechanical stability of a minimal RNA kissing complex elucidated by nonequilibrium molecular dynamics simulations.

An RNA kissing loop from the Moloney murine leukemia virus (MMLV) exhibits unusual mechanical stability despite having only two intermolecular base pairs. Mutations at this junction have been shown to destabilize genome dimerization, with concomitant reductions in viral packaging efficiency and infectivity. Optical tweezers experiments have shown that it requires as much force to break the MMLV kissing-loop complex as is required to unfold an 11-bp RNA hairpin [Li PTX, Bustamante C, Tinoco I (2006) Proc Natl Acad Sci USA 103:15847-15852]. Using nonequilibrium all-atom molecular dynamics simulations, we have developed a detailed model for the kinetic intermediates of the force-induced dissociation of the MMLV dimerization initiation site kissing loop. Two hundred and eight dissociation events were simulated (approximately 16 µs total simulation time) under conditions of constant applied external force, which we use to construct a Markov state model for kissing-loop dissociation. We find that the complex undergoes a conformational rearrangement, which allows for equal distribution of the applied force among all of the intermolecular hydrogen bonds, which is intrinsically more stable than the sequential unzipping of an ordinary hairpin. Stacking interactions with adjacent, unpaired loop adenines further stabilize the complex by increasing the repair rate of partially broken H-bonds. These stacking interactions are prominently featured in the transition state, which requires additional coordinates orthogonal to the end-to-end extension to be uniquely identified. We propose that these stabilizing features explain the unusual stability of other retroviral kissing-loop complexes such as the HIV dimerization site.

Cavities determine the pressure unfolding of proteins.

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules.

Guanidinium-rich molecules, such as cell-penetrating peptides, efficiently enter living cells in a non-endocytic energy-independent manner and transport a wide range of cargos, including drugs and biomarkers. The mechanism by which these highly cationic molecules efficiently cross the hydrophobic barrier imposed by the plasma membrane remains a fundamental open question. Here, a combination of computational results and in vitro and live-cell experimental evidence reveals an efficient energy-independent translocation mechanism for arginine-rich molecules. This mechanism unveils the essential role of guanidinium groups and two universal cell components: fatty acids and the cell membrane pH gradient. Deprotonated fatty acids in contact with the cell exterior interact with guanidinium groups, leading to a transient membrane channel that facilitates the transport of arginine-rich peptides toward the cell interior. On the cytosolic side, the fatty acids become protonated, releasing the peptides and resealing the channel. This fundamental mechanism appears to be universal across cells from different species and kingdoms.

Cosolvent effects on protein stability.

Proteins are marginally stable, and the folding/unfolding equilibrium of proteins in aqueous solution can easily be altered by the addition of small organic molecules known as cosolvents. Cosolvents that shift the equilibrium toward the unfolded ensemble are termed denaturants, whereas those that favor the folded ensemble are known as protecting osmolytes. Urea is a widely used denaturant in protein folding studies, and the molecular mechanism of its action has been vigorously debated in the literature. Here we review recent experimental as well as computational studies that show an emerging consensus in this problem. Urea has been shown to denature proteins through a direct mechanism, by interacting favorably with the peptide backbone as well as the amino acid side chains. In contrast, the molecular mechanism by which the naturally occurring protecting osmolyte trimethylamine N-oxide (TMAO) stabilizes proteins is not clear. Recent studies have established the strong interaction of TMAO with water. Detailed molecular simulations, when used with force fields that incorporate these interactions, can provide insight into this problem. We present the development of a model for TMAO that is consistent with experimental observations and that provides physical insight into the role of cosolvent-cosolvent interaction in determining its preferential interaction with proteins.

Folding and unfolding thermodynamics of the TC10b Trp-cage miniprotein.

We examine the folding-unfolding of a variant of the Trp-cage, known as TC10b, and compare structural stability, dynamics, and thermodynamics with that of the TC5b variant, using replica exchange molecular dynamics (REMD). The TC10b variant was designed to have larger helical stability by the substitution of amino acids with greater alpha helical propensities in the N-terminal region. Experiments have shown TC10b to possess larger overall stability than TC5b. Simulations starting from unbiased, unfolded initial conditions are run for 1 µs per replica. The calculations show a higher melting temperature for TC10b than TC5b, and suggest a more ordered folded structure through the elimination of a substate found in the folded ensemble of TC5b. We model the difference in Gibbs free energy, ΔG(P,T), of folding using the bootstrap statistical method, which is used to calculate uncertainties associated with the thermodynamic parameters for both variants of the Trp-cage. We find that while the shape of the area for which the protein is stability folded is elliptical for TC5b, there is a degree of uncertainty associated with that of TC10b, with one model suggesting elliptical and another suggesting hyperbolic. This model suggests that at high pressures, TC5b can experience pressure denaturation, but TC10b may not.

Equilibrium thermodynamics and folding kinetics of a short, fast-folding, beta-hairpin.

Equilibrium thermodynamics of a short beta-hairpin are studied using unbiased all-atom replica exchange molecular dynamics simulations in explicit solvent. An exploratory analysis of the free energy landscape of the system is provided in terms of various structural characteristics, for both the folded and unfolded ensembles. We find that the favorable interactions between the ends introduced by the tryptophan cap, along with the flexibility of the turn region, explain the remarkable stability of the folded state. Charging of the N termini results in effective roughening of the free energy landscape and stabilization of non-native contacts. Folding-unfolding dynamics are further discussed using a set of 2413 independent molecular dynamics simulations, 2 ns to 20 ns long, at the melting temperature of the beta-hairpin. A novel method for the construction of Markov models consisting of an iterative refinement of the discretization in reduced dimensionality is presented and used to generate a detailed kinetic network of the system. The hairpin is found to fold heterogeneously on sub-microsecond timescales, with the relative position of the tryptophan side chains driving the selection of the specific pathway.

Effects of truncating van der Waals interactions in lipid bilayer simulations.

In membrane simulations, it is known that truncating electrostatic interactions results in artificial ordering of lipids at the truncation distance. However, less attention has been paid to the effect of truncating van der Waals (VDW) interactions. Since the VDW potential decays as r(-6), it is frequently neglected beyond a cutoff of around 1 nm. In some cases, analytical dispersion corrections appropriate for isotropic systems are applied to the pressure and the potential energy. In this work, we systematically study the effect of truncating VDW interactions at different cutoffs in 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine bilayers with the Berger force field. We show that the area per lipid decreases systematically when the VDW cutoff (r(c)) increases. This dependence persists even when dispersion corrections are applied. Since the analytical form of the dispersion correction is only appropriate for isotropic systems, we suggest that a long VDW cutoff should be used in preference over a short VDW cutoff. To determine the appropriate cutoff, we simulate liquid pentadecane with the Berger parameters and find that r(c) ≥ 1.4 nm is sufficient to reproduce the density and the heat of vaporization of pentadecane. Bilayers simulated with r(c) ≥ 1.4 nm show an improved agreement with experiments in both the form factors and the deuterium order parameters. Finally, we report that the VDW cutoff has a significant impact on the lipid flip-flop energetics and an inappropriate short VDW cutoff results in a bilayer that is prone to form water defects across the bilayer.