Magnesium controls aptamer-expression platform switching in the SAM-I riboswitch.

Investigations of most riboswitches remain confined to the ligand-binding aptamer domain. However, during the riboswitch mediated transcription regulation process, the aptamer domain and the expression platform compete for a shared strand. If the expression platform dominates, an anti-terminator helix is formed, and the transcription process is active (ON state). When the aptamer dominates, transcription is terminated (OFF state). Here, we use an expression platform switching experimental assay and structure-based electrostatic simulations to investigate this ON-OFF transition of the full length SAM-I riboswitch and its magnesium concentration dependence. Interestingly, we find the ratio of the OFF population to the ON population to vary non-monotonically as magnesium concentration increases. Upon addition of magnesium, the aptamer domain pre-organizes, populating the OFF state, but only up to an intermediate magnesium concentration level. Higher magnesium concentration preferentially stabilizes the anti-terminator helix, populating the ON state, relatively destabilizing the OFF state. Magnesium mediated aptamer-expression platform domain closure explains this relative destabilization of the OFF state at higher magnesium concentration. Our study reveals the functional potential of magnesium in controlling transcription of its downstream genes and underscores the importance of a narrow concentration regime near the physiological magnesium concentration ranges, striking a balance between the OFF and ON states in bacterial gene regulation.

Structure-Based Model of RNA Pseudoknot Captures Magnesium-Dependent Folding Thermodynamics.

We develop a simple, coarse-grained approach for simulating the folding of the Beet Western Yellow Virus (BWYV) pseudoknot toward the goal of creating a transferable model that can be used to study other small RNA molecules. This approach combines a structure-based model (SBM) of RNA with an electrostatic scheme that has previously been shown to correctly reproduce ionic condensation in the native basin. Mg2+ ions are represented explicitly, directly incorporating ion-ion correlations into the system, and K+ is represented implicitly, through the mean-field generalized Manning counterion condensation theory. Combining the electrostatic scheme with a SBM enables the electrostatic scheme to be tested beyond the native basin. We calibrate the SBM to reproduce experimental BWYV unfolding data by eliminating overstabilizing backbone interactions from the molecular contact map and by strengthening base pairing and stacking contacts relative to other native contacts, consistent with the experimental observation that relative helical stabilities are central determinants of the RNA unfolding sequence. We find that this approach quantitatively captures the Mg2+ dependence of the folding temperature and generates intermediate states that better approximate those revealed by experiment. Finally, we examine how our model captures Mg2+ condensation about the BWYV pseudoknot and a U-tail variant, for which the nine 3' end nucleotides are replaced with uracils, and find our results to be consistent with experimental condensation measurements. This approach can be easily transferred to other RNA molecules by eliminating and strengthening the same classes of contacts in the SBM and including generalized Manning counterion condensation.

RNA as a Complex Polymer with Coupled Dynamics of Ions and Water in the Outer Solvation Sphere.

We unravel the internal and collective modes of a widely studied 58-nucleotide rRNA fragment in solvent using atomically detailed molecular dynamics simulations. The variation of lifetimes for water hydrogen bonds with nucleotide groups indicates heterogeneity of water dynamics on the RNA surface. The time scales of interactions of the discrete water molecules with RNA nucleotides extend from several hundred picoseconds to a few nanoseconds. We determine all of the association sites and the spatial distribution of residence times for Mg2+, K+, and water molecules in those sites. We provide insights into the population of Mg2+ and K+ ions and water molecules in the outer sphere and how their fluctuations are intricately linked with the kinetics of the 58-mer. We find that many of the long-lived Mg2+ sites identified from the simulations agree with the locations of ions in the X-ray structure. We determine the excess ion atmosphere around the rRNA and compare it with experimental data. We investigate the collective behavior of RNA, ions, and water, by performing a joint principle component analysis for the Cartesian coordinates of the RNA phosphorus atoms and for the occupation counts of the association sites. Our results indicate that the 58-mer system is a complex polymer, composed of RNA that is encased by a fluctuating network of associated counterions, co-ions, and water.

Uncovering the molecular mechanisms behind disease-associated leptin variants.

The pleiotropic hormone leptin has a pivotal role in regulating energy balance by inhibiting hunger and increasing energy expenditure. Homozygous mutations found in the leptin gene are associated with extreme obesity, marked hyperphagia, and impaired immune function. Although these mutations have been characterized in vivo, a detailed understanding of how they affect leptin structure and function remains elusive. In the current work, we used NMR, differential scanning calorimetry, molecular dynamics simulations, and bioinformatics calculations to characterize the effects of these mutations on leptin structure and function and binding to its cognate receptor. We found that mutations identified in patients with congenital leptin deficiency not only cause leptin misfolding or aggregation, but also cause changes in the dynamics of leptin residues on the receptor-binding interface. Therefore, we infer that mutation-induced leptin deficiency may arise from several distinct mechanisms including (i) blockade of leptin receptor interface II, (ii) decreased affinity in the second step of leptin's interaction with its receptor, (iii) leptin destabilization, and (iv) unsuccessful threading through the covalent loop, leading to leptin misfolding/aggregation. We propose that this expanded framework for understanding the mechanisms underlying leptin deficiency arising from genetic mutations may be useful in designing therapeutics for leptin-associated disorders.

Fluorescent Proteins Detect Host Structural Rearrangements via Electrostatic Mechanism.

The rational design of genetically encoded fluorescent biosensors, which can detect rearrangements of target proteins via interdomain allostery, is hindered by the absence of mechanistic understanding of the underlying photophysics. Here, we focus on the modulation of fluorescence by mechanical perturbation in a popular biological probe: enhanced Green Fluorescent Protein (eGFP). Using a combination of molecular dynamics (MD) simulations and quantum chemistry, and a set of physically motivated assumptions, we construct a map of fluorescence quantum yield as a function of a 2D electric field imposed by the protein environment on the fluorophore. This map is transferable between Tsien's Class 2 GFP's, and it allows one to estimate the shifts in fluorescence intensity due to mechanical perturbations directly from MD simulations. We use it in combination with steered MD simulations to put forward a hypothesis for the mechanism of a genetically encoded voltage probe (ArcLight) whose mechanism is currently under debate.

Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells.

The NEET family is a relatively new class of three related [2Fe-2S] proteins (CISD1-3), important in human health and disease. While there has been growing interest in the homodimeric gene products of CISD1 (mitoNEET) and CISD2 (NAF-1), the importance of the inner mitochondrial CISD3 protein has only recently been recognized in cancer. The CISD3 gene encodes for a monomeric protein that contains two [2Fe-2S] CDGSH motifs, which we term mitochondrial inner NEET protein (MiNT). It folds with a pseudosymmetrical fold that provides a hydrophobic motif on one side and a relatively hydrophilic surface on the diametrically opposed surface. Interestingly, as shown by molecular dynamics simulation, the protein displays distinct asymmetrical backbone motions, unlike its homodimeric counterparts that face the cytosolic side of the outer mitochondrial membrane/endoplasmic reticulum (ER). However, like its counterparts, our biological studies indicate that knockdown of MiNT leads to increased accumulation of mitochondrial labile iron, as well as increased mitochondrial reactive oxygen production. Taken together, our study suggests that the MiNT protein functions in the same pathway as its homodimeric counterparts (mitoNEET and NAF-1), and could be a key player in this pathway within the mitochondria. As such, it represents a target for anticancer or antidiabetic drug development.

A magnesium-induced triplex pre-organizes the SAM-II riboswitch.

Our 13C- and 1H-chemical exchange saturation transfer (CEST) experiments previously revealed a dynamic exchange between partially closed and open conformations of the SAM-II riboswitch in the absence of ligand. Here, all-atom structure-based molecular simulations, with the electrostatic effects of Manning counter-ion condensation and explicit magnesium ions are employed to calculate the folding free energy landscape of the SAM-II riboswitch. We use this analysis to predict that magnesium ions remodel the landscape, shifting the equilibrium away from the extended, partially unfolded state towards a compact, pre-organized conformation that resembles the ligand-bound state. Our CEST and SAXS experiments, at different magnesium ion concentrations, quantitatively confirm our simulation results, demonstrating that magnesium ions induce collapse and pre-organization. Agreement between theory and experiment bolsters microscopic interpretation of our simulations, which shows that triplex formation between helix P2b and loop L1 is highly sensitive to magnesium and plays a key role in pre-organization. Pre-organization of the SAM-II riboswitch allows rapid detection of ligand with high selectivity, which is important for biological function.

SMOG 2: A Versatile Software Package for Generating Structure-Based Models.

Molecular dynamics simulations with coarse-grained or simplified Hamiltonians have proven to be an effective means of capturing the functionally important long-time and large-length scale motions of proteins and RNAs. Originally developed in the context of protein folding, structure-based models (SBMs) have since been extended to probe a diverse range of biomolecular processes, spanning from protein and RNA folding to functional transitions in molecular machines. The hallmark feature of a structure-based model is that part, or all, of the potential energy function is defined by a known structure. Within this general class of models, there exist many possible variations in resolution and energetic composition. SMOG 2 is a downloadable software package that reads user-designated structural information and user-defined energy definitions, in order to produce the files necessary to use SBMs with high performance molecular dynamics packages: GROMACS and NAMD. SMOG 2 is bundled with XML-formatted template files that define commonly used SBMs, and it can process template files that are altered according to the needs of each user. This computational infrastructure also allows for experimental or bioinformatics-derived restraints or novel structural features to be included, e.g. novel ligands, prosthetic groups and post-translational/transcriptional modifications. The code and user guide can be downloaded at http://smog-server.org/smog2.

Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures.

The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein's functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism.

Pierced Lasso Bundles are a new class of knot-like motifs.

A four-helix bundle is a well-characterized motif often used as a target for designed pharmaceutical therapeutics and nutritional supplements. Recently, we discovered a new structural complexity within this motif created by a disulphide bridge in the long-chain helical bundle cytokine leptin. When oxidized, leptin contains a disulphide bridge creating a covalent-loop through which part of the polypeptide chain is threaded (as seen in knotted proteins). We explored whether other proteins contain a similar intriguing knot-like structure as in leptin and discovered 11 structurally homologous proteins in the PDB. We call this new helical family class the Pierced Lasso Bundle (PLB) and the knot-like threaded structural motif a Pierced Lasso (PL). In the current study, we use structure-based simulation to investigate the threading/folding mechanisms for all the PLBs along with three unthreaded homologs as the covalent loop (or lasso) in leptin is important in folding dynamics and activity. We find that the presence of a small covalent loop leads to a mechanism where structural elements slipknot to thread through the covalent loop. Larger loops use a piercing mechanism where the free terminal plugs through the covalent loop. Remarkably, the position of the loop as well as its size influences the native state dynamics, which can impact receptor binding and biological activity. This previously unrecognized complexity of knot-like proteins within the helical bundle family comprises a completely new class within the knot family, and the hidden complexity we unraveled in the PLBs is expected to be found in other protein structures outside the four-helix bundles. The insights gained here provide critical new elements for future investigation of this emerging class of proteins, where function and the energetic landscape can be controlled by hidden topology, and should be take into account in ab initio predictions of newly identified protein targets.

Altered backbone and side-chain interactions result in route heterogeneity during the folding of interleukin-1ß (IL-1ß).

Deletion of the ß-bulge trigger-loop results in both a switch in the preferred folding route, from the functional loop packing folding route to barrel closure, as well as conversion of the agonist activity of IL-1ß into antagonist activity. Conversely, circular permutations of IL-1ß conserve the functional folding route as well as the agonist activity. These two extremes in the folding-functional interplay beg the question of whether mutations in IL-1ß would result in changes in the populations of heterogeneous folding routes and the signaling activity. A series of topologically equivalent water-mediated ß-strand bridging interactions within the pseudosymmetric ß-trefoil fold of IL-1ß highlight the backbone water interactions that stabilize the secondary and tertiary structure of the protein. Additionally, conserved aromatic residues lining the central cavity appear to be essential for both stability and folding. Here, we probe these protein backbone-water molecule and side chain-side chain interactions and the role they play in the folding mechanism of this geometrically stressed molecule. We used folding simulations with structure-based models, as well as a series of folding kinetic experiments to examine the effects of the F42W core mutation on the folding landscape of IL-1ß. This mutation alters water-mediated backbone interactions essential for maintaining the trefoil fold. Our results clearly indicate that this perturbation in the primary structure alters a structural water interaction and consequently modulates the population of folding routes accessed during folding and signaling activity.

The dominant folding route minimizes backbone distortion in SH3.

Energetic frustration in protein folding is minimized by evolution to create a smooth and robust energy landscape. As a result the geometry of the native structure provides key constraints that shape protein folding mechanisms. Chain connectivity in particular has been identified as an essential component for realistic behavior of protein folding models. We study the quantitative balance of energetic and geometrical influences on the folding of SH3 in a structure-based model with minimal energetic frustration. A decomposition of the two-dimensional free energy landscape for the folding reaction into relevant energy and entropy contributions reveals that the entropy of the chain is not responsible for the folding mechanism. Instead the preferred folding route through the transition state arises from a cooperative energetic effect. Off-pathway structures are penalized by excess distortion in local backbone configurations and contact pair distances. This energy cost is a new ingredient in the malleable balance of interactions that controls the choice of routes during protein folding.

The role of atomic level steric effects and attractive forces in protein folding.

Protein folding into tertiary structures is controlled by an interplay of attractive contact interactions and steric effects. We investigate the balance between these contributions using structure-based models using an all-atom representation of the structure combined with a coarse-grained contact potential. Tertiary contact interactions between atoms are collected into a single broad attractive well between the C(ß) atoms between each residue pair in a native contact. Through the width of these contact potentials we control their tolerance for deviations from the ideal structure and the spatial range of attractive interactions. In the compact native state dominant packing constraints limit the effects of a coarse-grained contact potential. During folding, however, the broad attractive potentials allow an early collapse that starts before the native local structure is completely adopted. As a consequence the folding transition is broadened and the free energy barrier is decreased. Eventually two-state folding behavior is lost completely for systems with very broad attractive potentials. The stabilization of native-like residue interactions in non-perfect geometries early in the folding process frequently leads to structural traps. Global mirror images are a notable example. These traps are penalized by the details of the repulsive interactions only after further collapse. Successful folding to the native state requires simultaneous guidance from both attractive and repulsive interactions.

Allostery in the ferredoxin protein motif does not involve a conformational switch.

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.

Simplified interpretation of transport in disordered inorganic ion conductors from vacancy dynamics.

Ion transport in structurally disordered inorganic ion conductors can be interpreted as cation jumps between sites provided by the network. Because of the small number of vacant sites and strong intercationic Coulomb interaction, their dynamics is very complex. Based on molecular dynamics simulations we recast the ion dynamics via a sophisticated mapping procedure into the corresponding vacancy dynamics. Remarkably, in this framework, the transport can be interpreted to a very good approximation as a noninteracting single-particle processes. In particular, the macroscopic conductivity can be directly obtained from the local vacancy hopping rates.

The cationic energy landscape in alkali silicate glasses: Properties and relevance.

Individual cationic site energies are explicitly determined from molecular dynamics simulations of alkali silicate glasses, and the properties and relevance of this local energetics to ion transport are studied. The absence of relaxations on the time scale of ion transport proves the validity of a static description of the energy landscape, as it is generally used in hopping models. The Coulomb interaction among the cations turns out to be essential to obtain an average energy landscape in agreement with typical simplified hopping models. Strong correlations exist both between neighboring sites and between different energetic contributions at one site, and they shape essential characteristics of the energy landscape. A model energy landscape with a single vacancy is used to demonstrate why average site energies, including the full Coulomb interaction, are still insufficient to describe the site population of ions, or their dynamics. This model explains how the relationship between energetics and ion dynamics is weakened, and thus establishes conclusively that a hopping picture with static energies fails to capture all the relevant information. It is therefore suggested that alternative simplified models of ion conduction are needed.

Robustness and generalization of structure-based models for protein folding and function.

Functional dynamics of native proteins share the energy landscape that guides folding into the native state. Folding simulations of structure-based protein models, using an minimally frustrated energy landscape dominated by native interactions, can describe the geometrical aspects of the folding mechanism. Technical limitations imposed by the fixed shape of conventional contact potentials are a key obstacle toward advanced applications of structure-based models like allostery or ligand binding, which require multiple stable conformations. Generalizations of existing models, commonly using Lennard-Jones-like potentials, lead to inevitable clashes between their repulsive branches. To resolve these challenges, a new contact potential is developed that combines an attractive part based on Gaussians with a separate repulsive term allowing flexibility for adjustments of the potential shape. With this new model multiple minima for studies of functional transitions can be introduced easily and consistently. A sensitivity analysis for five small proteins confirms the robust behavior of structure-based models with our adaptable potential and explores their capacity for quantitative adjustment of the folding thermodynamics. We demonstrate its ability to incorporate alternative contact distances in simulations of structural transitions for the well-studied ROP dimer. Individual contact pairs can switch between distinct states to match the competing syn and anti structures. The flexibility of the new potential facilitates advanced uses of structure-based models. Depending on the application, features can be chosen from physical considerations or to match experiments. Generalized models can be built from multiple structures to study structural transitions or effects of disorder.

Complete identification of alkali sites in ion conducting lithium silicate glasses: a computer study of ion dynamics.

The available sites for ions in a typical disordered ionic conductor are determined. For this purpose we devised a straightforward algorithm which via cluster analysis identifies these sites from long time ionic trajectories below the glass transition. This is exemplified for a lithium silicate glass (Li2O)(x)(SiO2)((1-x)) for x=0.5 and x=0.1. We find for both concentrations that the number of sites is only slightly bigger than the number of ions. This result suggests a theoretical description of the dynamics in terms of mobile vacancies as most appropriate. Furthermore, identification of the ionic sites allows one to obtain detailed characteristics of the ionic motion, e.g., quantification of correlated forward-backward jumps.