Atomic Detail Research Articles

Curvature Footprints of Transmembrane Proteins in Simulations with the Martini Force Field.

Membranes play essential roles in biological systems and are tremendously diverse in the topologies and chemical and elastic properties that define their functions. In many cases, a given membrane may display considerable heterogeneity, with localized clusters of lipids and proteins exhibiting distinct characteristics compared to adjoining regions. These lipid-protein assemblies can span nanometers to micrometers and are associated with cellular processes such as transport and signaling. While lipid-protein assemblages are dynamic, they can be stabilized by coupling between local membrane composition and shape. Due to the inherent difficulty in resolving atomistic details of membrane proteins in their native lipid environments, these complexes are notoriously challenging to study experimentally; however, molecular dynamics (MD) simulations might be a viable alternative. Here, we aim to assess the utility of coarse-grained (CG) MD simulations with the Martini force field for studying membrane curvature induced by transmembrane (TM) proteins that are reported to generate local curvature. The direction and magnitude of curvature induced by five different TM proteins, as well as certain lipid-protein and protein-protein interactions, were found to be in good agreement with available reference data.

Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS/FTD.

The abnormal GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause the fatal neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. The transcribed RNA HREs, short for r(G4C2)n, can form toxic RNA foci which sequestrate RNA binding proteins and impair RNA processing, ultimately leading to neurodegeneration. Here, we determined the crystal structure of r(G4C2)2, which folds into a parallel tetrameric G-quadruplex composed of two four-layer dimeric G-quadruplex via 5'-to-5' stacking in coordination with a K+ ion. Notably, the two C bases locate at 3'- end stack on the outer G-tetrad with the assistance of two additional K+ ions. The high-resolution structure reported here lays a foundation in understanding the mechanism of neurological toxicity of RNA HREs. Furthermore, the atomic details provide a structural basis for the development of potential therapeutic agents against the fatal neurodegenerative diseases ALS/FTD.

Metalloid transporters in plants: bridging the gap in molecular structure and physiological exaptation

Abstract The rhizosphere contains both essential nutrients and potentially harmful substances for plant growth. Plants, as sessile organisms, must efficiently absorb the necessary nutrients while actively avoiding the uptake of toxic compounds. Metalloids, elements that exhibit properties of both metals and non-metals, can have different effects on plant growth, from being essential and beneficial to being toxic. This toxicity arises due to either the dosage of exposure or the specific elemental type. To utilize or detoxify these elements, plants have developed various transporters regulating their uptake and distribution in plants. Genomic sequence analysis suggests that such transporter families exist throughout the plant kingdom, from chlorophytes to higher plants. These transporters form defined families with related transport preferences. The isoforms within these families have evolved with specialized functions regulated by defined selectivity. Hence, understanding the chemistry of transporters to atomic detail is important to achieve the desired genetic modifications for crop improvement. We outline various adaptations in plant transport systems to deal with metalloids, including their uptake, distribution, detoxification, and homeostasis in plant tissues. Structural parallels are drawn to other nutrient transporter systems to support emerging themes of functional diversity of active sites of transporters, elucidating plant adaptations to utilize and extrude metalloid concentrations. Considering the observed physiological importance of metalloids, this review highlights the shared and disparate features in metalloid transport systems and their corresponding nutrient transporters.

Effects of Phosphorylation on Protein Backbone Dynamics and Conformational Preferences.

Phosphorylations are the most common and extensively studied post-translational modification (PTM) of proteins in eukaryotes. They constitute a major regulatory mechanism, modulating protein function, protein-protein interactions, as well as subcellular localization. Phosphorylation sites are preferably located in intrinsically disordered regions and have been shown to trigger structural rearrangements and order-to-disorder transitions. They can therefore have a significant effect on protein backbone dynamics or conformation, but only sparse experimental data are available. To obtain a more general description of how and when phosphorylations have a significant effect on protein behavior, molecular dynamics (MD) currently provides the only suitable framework to study these effects at a large scale in atomistic detail. This study develops a systematic MD simulation framework to explore the influence of phosphorylations on the local backbone dynamics and conformational propensities of proteins. Through a series of glycine-backbone peptides, we studied the effects of amino acid residues including the three most common phosphorylations (Ser, Thr, and Tyr), on local backbone dynamics and conformational propensities. We further extended our study to investigate the interactions of all such residues between position i to positions i + 1, i + 2, i + 3, and i + 4 in such peptides. The final data set comprises structural ensembles for 3393 sequences with more than 1 μs of sampling for each ensemble. To validate the relevance of the results, the structural and conformational properties extracted from the MD simulations are compared to NMR data from the Biological Magnetic Resonance Data Bank. The systematic nature of this study enables the projection of the gained knowledge onto any phosphorylation site in the proteome and provides a general framework for the study of further PTMs. The full data set is publicly available, as a training and reference set.

Molecular storytelling: a conceptual framework for teaching and learning with molecular case studies

Molecular case studies (MCSs) provide educational opportunities to explore biomolecular structure and function using data from public bioinformatics resources. The conceptual basis for the design of MCSs has yet to be fully discussed in the literature, so we present molecular storytelling as a conceptual framework for teaching with case studies. Whether the case study aims to understand the biology of a specific disease and design its treatments or track the evolution of a biosynthetic pathway, vast amounts of structural and functional data, freely available in public bioinformatics resources, can facilitate rich explorations in atomic detail. To help biology and chemistry educators use these resources for instruction, a community of scholars collaborated to create the Molecular CaseNet. This community uses storytelling to explore biomolecular structure and function while teaching biology and chemistry. In this article, we define the structure of an MCS and present an example. Then, we articulate the evolution of a conceptual framework for developing and using MCSs. Finally, we related our framework to the development of technological, pedagogical, and content knowledge (TPCK) for educators in the Molecular CaseNet. The report conceptualizes an interdisciplinary framework for teaching about the molecular world and informs lesson design and education research.

Decoding the κ Opioid Receptor (KOR): Advancements in Structural Understanding and Implications for Opioid Analgesic Development.

The opioid crisis in the United States is a significant public health issue, with a nearly threefold increase in opioid-related fatalities between 1999 and 2014. In response to this crisis, society has made numerous efforts to mitigate its impact. Recent advancements in understanding the structural intricacies of the κ opioid receptor (KOR) have improved our knowledge of how opioids interact with their receptors, triggering downstream signaling pathways that lead to pain relief. This review concentrates on the KOR, offering crucial structural insights into the binding mechanisms of both agonists and antagonists to the receptor. Through comparative analysis of the atomic details of the binding site, distinct interactions specific to agonists and antagonists have been identified. These insights not only enhance our understanding of ligand binding mechanisms but also shed light on potential pathways for developing new opioid analgesics with an improved risk-benefit profile.

Na+/K+-ATPase: More than an Electrogenic Pump.

The sodium pump, or Na+/K+-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na+) and potassium (K+) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA's role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell-cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA β-subunits as cell adhesion molecules in glia and epithelial cells.

Thrombin - A Molecular Dynamics Perspective.

Thrombin is a crucial enzyme involved in blood coagulation, essential for maintaining circulatory system integrity and preventing excessive bleeding. However, thrombin is also implicated in pathological conditions such as thrombosis and cancer. Despite the application of various experimental techniques, including X-ray crystallography, NMR spectroscopy, and HDXMS, none of these methods can precisely detect thrombin's dynamics and conformational ensembles at high spatial and temporal resolution. Fortunately, molecular dynamics (MD) simulation, a computational technique that allows the investigation of molecular functions and dynamics in atomic detail, can be used to explore thrombin behavior. This review summarizes recent MD simulation studies on thrombin and its interactions with other biomolecules. Specifically, the 17 studies discussed here provide insights into thrombin's switch between 'slow' and 'fast' forms, active and inactive forms, the role of Na+ binding, the effects of light chain mutation, and thrombin's interactions with other biomolecules. The findings of these studies have significant implications for developing new therapies for thrombosis and cancer. By understanding thrombin's complex behavior, researchers can design more effective drugs and treatments that target thrombin.

High-resolution in situ structures of mammalian respiratory supercomplexes

Mitochondria play a pivotal part in ATP energy production through oxidative phosphorylation, which occurs within the inner membrane through a series of respiratory complexes1–4. Despite extensive in vitro structural studies, determining the atomic details of their molecular mechanisms in physiological states remains a major challenge, primarily because of loss of the native environment during purification. Here we directly image porcine mitochondria using an in situ cryo-electron microscopy approach. This enables us to determine the structures of various high-order assemblies of respiratory supercomplexes in their native states. We identify four main supercomplex organizations: I1III2IV1, I1III2IV2, I2III2IV2 and I2III4IV2, which potentially expand into higher-order arrays on the inner membranes. These diverse supercomplexes are largely formed by ‘protein–lipids–protein’ interactions, which in turn have a substantial impact on the local geometry of the surrounding membranes. Our in situ structures also capture numerous reactive intermediates within these respiratory supercomplexes, shedding light on the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. Structural comparison of supercomplexes from mitochondria treated under different conditions indicates a possible correlation between conformational states of complexes I and III, probably in response to environmental changes. By preserving the native membrane environment, our approach enables structural studies of mitochondrial respiratory supercomplexes in reaction at high resolution across multiple scales, from atomic-level details to the broader subcellular context.

High Pressure Promotes Binding of the Allosteric Inhibitor Zn2+-Cyclen in Crystals of Activated H-Ras.

In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+-cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+-cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+-cyclen binding is observed at 500 MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+-cyclen to H-Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x-ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low-populated conformations at ambient conditions, enabling the design of specific inhibitors.

Revealing the Effect of Stereocontrol on Intermolecular Interactions between Abiotic, Sequence-Defined Polyurethanes and a Ligand.

The development of precision polymer synthesis has facilitated access to a diverse library of abiotic structures wherein chiral monomers are positioned at specific locations within macromolecular chains. These structures are anticipated to exhibit folding characteristics similar to those of biotic macromolecules and possess comparable functionalities. However, the extensive sequence space and numerous variables make selecting a sequence with the desired function challenging. Therefore, revealing sequence-function dependencies and developing practical tools are necessary to analyze their conformations and molecular interactions. In this study, we investigate the effect of stereochemistry, which dictates the spatial location of backbone and pendant groups, on the interaction between sequence-defined oligourethanes and bisphenol A ligands. Various methods are explored to analyze the receptor-like properties of model oligomers and the ligand. The accuracy of molecular dynamics simulations and experimental techniques is assessed to uncover the impact of discrete changes in stereochemical arrangements on the structures of the resulting complexes and their binding strengths. Detailed computational investigations providing atomistic details show that the formed complexes demonstrate significant structural diversity depending on the sequence of stereocenters, thus affecting the oligomer-ligand binding strength. Among the tested techniques, the fluorescence spectroscopy data, fitted to the Stern-Volmer equation, are consistently aligned with the calculations, thus validating the developed simulation methodology. The developed methodology opens a way to engineer the structure of sequence-defined oligomers with receptor-like functionality to explore their practical applications, e.g., as sensory materials.

Rapid prediction of grain boundary network evolution in nanomaterials utilizing a generative machine learning approach

Predicting the behavior of nanomaterials under various conditions presents a significant challenge due to their complex microstructures. While high-fidelity modeling techniques, such as molecular dynamics (MD) simulations, are effective, they are also computationally demanding. Machine learning (ML) models have opened new avenues for the rapid exploration of design spaces. In this work, we developed a deep learning framework based on a conditional generative adversarial network (cGAN) to predict the evolution of grain boundary (GB) networks in nanocrystalline materials under mechanical loads, incorporating both morphological and atomic details. We conducted MD simulations on nanocrystalline tungsten and used the resulting ground-truth data to train our cGAN model. We assessed the performance of our cGAN model by comparing it to a Convolutional Autoencoder (ConvAE) model and examining the impact of changes in geometric morphology and loading conditions on the model's performance. Our cGAN model demonstrated high accuracy in predicting GB network evolution under a variety of conditions. This developed framework shows potential for predicting various materials' behaviors across a wide range of nanomaterials.

Atomistic Insights into Nucleation of Ferroelastic Domains in PbTiO3.

Understanding the nucleation mechanism of domains is essential for domain engineering of perovskite ferroelectric materials. We proposed and examined atomistic details for nucleating ferroelastic (FS) domains by integrating topological analysis and first-principles calculations. FS domains are crystallographically treated as deformation twins. The conventional shear-shuffle nucleation mechanism under simple shear deformation is ruled out because the 1-layer elementary twinning disconnection (TD) cannot nucleate and glide in a perfect matrix. Thus, the pure-shuffle nucleation mechanism under pure shear deformation is proposed due to kinetically favored atomic shuffling. The coherency stress associated with the coherent nucleus is relaxed via forming misfit dislocations, accompanied by formation and sharpening of diffused (110)m∥(110)d domain walls (DWs). The sharp DWs enable growth of the FS nucleus through successive nucleation and gliding of TDs. These findings enrich the knowledge of domain behavior in perovskite ferroelectric materials.

Atomistic Details of Methyl Linoleate Pyrolysis: Direct Molecular Dynamics Simulation of Converting Biodiesel to Petroleum Products

Dependence on petroleum and petrochemical products is unsustainable; it is both a finite resource and an environmental hazard. Biodiesel has many attractive qualities, including a sustainable feedstock; however, it has its complications. The pyrolysis (a process already in common use in the petroleum industry) of biodiesel has demonstrated the formation of smaller hydrocarbons comprising many petrochemical products but experiments suffer from difficulty quantifying the myriad reaction pathways followed and products formed. A computational simulation of pyrolysis using “ab initio molecular dynamics” offers atomic-level detail of the reaction pathways and products formed. Herein, the most prevalent fatty-acid ester (methyl linoleate) from the most prevalent feedstock for biodiesel in the United States (soybean oil) is studied. Temperature acceleration within the atom-centered density matrix propagation formalism (Car–Parrinello) utilizing the D3-M06-2X/6-31+G(d,p) model chemistry is used to compose an ensemble of trajectories. The results are grounded in comparison to experimental studies through agreement in the following: (1) the extent of reactivity (40% in the experimental and 36.1% in this work), (2) the homology of hydrocarbon products formed (wt % of C6–C10 products), and (3) the CO/CO2 product ratio. Deoxygenation pathways are critically analyzed (as the presence of oxygen in biodiesel represents a disadvantage in its current use). Within this ensemble, deoxygenation was found to proceed through two subclasses: (1) spontaneous deoxygenation, following one of four possible pathways; or (2) induced deoxygenation, following one of three possible pathways.

Bridging the maytansine and vinca sites: Cryptophycins target β-tubulin’s T5-loop

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αβ-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of β-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship (SAR) studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of β-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "βT5-loop site” that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple β-tubulin binding sites.

Impact of an Ionic Liquid Solution on Horseradish Peroxidase Activity.

Horseradish peroxidase (HRP) is an enzyme that oxidizes pollutants from wastewater. A previous report indicated that peroxidases can have an enhancement in initial enzymatic activity in an aqueous solution of 0.26 M 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIm][EtSO4]) at neutral pH. However, the atomistic details remain elusive. In the enzymatic landscape of HRP, compound II (Cpd II) plays a key role and involves a histidine (H42) residue. Cpd II exists as oxoferryl (2a) or hydroxoferryl (2b(FeIV)) forms, where 2a is the predominantly observed form in experimental studies. Intriguingly, the ferric 2b(FeIII) form seen in synthetic complexes has not been observed in HRP. Here, we have investigated the structure and dynamics of HRP in pure water and aqueous [EMIm][EtSO4] (0.26 M), as well as the reaction mechanism of 2a to 2b conversion using polarizable molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations. When HRP is solvated in aq [EMIm][EtSO4], the catalytic water displaces, and H42 directly orients over the ferryl moiety, allowing a direct proton transfer (PT) with a significant energy barrier reduction. Conversely, in neat water, the reaction of 2a to 2b follows the previously reported mechanism. We further investigated the deprotonated form of H42. Analysis of the electric fields at the active site indicates that the aq [EMIm][EtSO4] medium facilitates the reaction by providing a more favorable environment compared with the system solvated in neat water. Overall, the atomic level supports the previous experimental observations and underscores the importance of favorable electric fields in the active site to promote catalysis.

Exploring the Interactions between two Ligands, UCB-J and UCB-F, and Synaptic Vesicle Glycoprotein 2 Isoforms.

In silico modeling was applied to study the efficiency of two ligands, namely, UCB-J and UCB-F, to bind to isoforms of the synaptic vesicle glycoprotein 2 (SV2) that are involved in the regulation of synaptic function in the nerve terminals, with the ultimate goal to understand the selectivity of the interaction between UCB-J and UCB-F to different isoforms of SV2. Docking and large-scale molecular dynamics simulations were carried out to unravel various binding patterns, types of interactions, and binding free energies, covering hydrogen bonding and nonspecific hydrophobic interactions, water bridge, π-π, and cation-π interactions. The overall preference for bonding types of UCB-J and UCB-F with particular residues in the protein pockets can be disclosed in detail. A unique interaction fingerprint, namely, hydrogen bonding with additional cation-π interaction with the pyridine moiety of UCB-J, could be established as an explanation for its high selectivity over the SV2 isoform A (SV2A). Other molecular details, primarily referring to the presence of π-π interactions and hydrogen bonding, could also be analyzed as sources of selectivity of the UCB-F tracer for the three isoforms. The simulations provide atomic details to support future development of new selective tracers targeting synaptic vesicle glycoproteins and their associated diseases.

NMR characterization of RNA binding property of the DEAD-box RNA helicase DDX3X and its implications for helicase activity

The DEAD-box RNA helicase (DDX) plays a central role in many aspects of RNA metabolism by remodeling the defined structure of RNA molecules. While a number of structural studies have revealed the atomistic details of the interaction between DDX and RNA ligands, the molecular mechanism of how this molecule unwinds a structured RNA into an unstructured single-stranded RNA (ssRNA) has largely remained elusive. This is due to challenges in structurally characterizing the unwinding intermediate state and the lack of thermodynamic details underlying this process. In this study, we use solution nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction of human DDX3X, a member of the DDX family, with various RNA ligands. Our results show that the inherent binding affinity of DDX3X for ssRNA is significantly higher than that for structured RNA elements. This preferential binding, accompanied by the formation of a domain-closed conformation in complex with ssRNA, effectively stabilizes the denatured ssRNA state and thus underlies the unwinding activity of DDX3X. Our results provide a thermodynamic and structural basis for the DDX function, whereby DDX can recognize and remodel a distinct set of structured RNAs to participate in a wide range of physiological processes.

Functional protein dynamics in a crystal.

Proteins are molecular machines and to understand how they work, we need to understand how they move. New pump-probe time-resolved X-ray diffraction methods open up ways to initiate and observe protein motions with atomistic detail in crystals on biologically relevant timescales. However, practical limitations of these experiments demands parallel development of effective molecular dynamics approaches to accelerate progress and extract meaning. Here, we establish robust and accurate methods for simulating dynamics in protein crystals, a nontrivial process requiring careful attention to equilibration, environmental composition, and choice of force fields. With more than seven milliseconds of sampling of a single chain, we identify critical factors controlling agreement between simulation and experiments and show that simulated motions recapitulate ligand-induced conformational changes. This work enables a virtuous cycle between simulation and experiments for visualizing and understanding the basic functional motions of proteins.

Interactions of NaCl with cellulose I$$\beta$$ crystal surfaces and the effect on cellulose hydration: a molecular dynamics study

AbstractCrystalline nanocellulose is widely used for example, in the paper-making and food industries, as support matrix material or reinforcement of polymer materials, but also in drug carrier and nanomedicine applications. Interestingly, aqueous solutions of cellulose are extremely sensitive to small amounts of added salt yet mere considerations of charge screening leave open questions regarding the mechanisms, especially for unmodified cellulose in aqueous solutions. Here, we map NaCl ion distributions and the effect of added NaCl salt on the hydration of I$$\beta$$ β cellulose nanocrystal (CNC) surfaces by atomistic detail molecular dynamics simulations with explicit water solvent. The simulations reveal the dependency of the hydration layers of the six surfaces of CNCs on the ions, as well as NaCl ion binding sites, and preferences in terms of binding free energy for the ions near CNC surfaces at different NaCl concentrations. We discuss the modelling results against our prior rheology characterization of cellulose solutions. Together, the results indicate that the high sensitivity of cellulose aqueous solutions to added salt rises from the ions near the surface changing locally the ordering and structure of the hydration layers of the CNC surfaces. The revealed mechanism of salt-induced viscosity changes in cellulose aqueous solutions allows advanced design of gelling CNC systems for various end uses and may also guide tuning cellulose interactions by different solvent environments.