Computational approaches to investigate fluoride binding, selectivity and transport across the membrane.

The use of molecular dynamics (MD) simulations to study biomolecular systems has proven reliable in elucidating atomic-level details of structure and function. In this chapter, MD simulations were used to uncover new insights into two phylogenetically unrelated bacterial fluoride (F-) exporters: the CLCF F-/H+ antiporter and the Fluc F- channel. The CLCF antiporter, a member of the broader CLC family, has previously revealed unique stoichiometry, anion-coordinating residues, and the absence of an internal glutamate crucial for proton import in the CLCs. Through MD simulations enhanced with umbrella sampling, we provide insights into the energetics and mechanism of the CLCF transport process, including its selectivity for F- over HF. In contrast, the Fluc F- channel presents a novel architecture as a dual topology dimer, featuring two pores for F- export and a central non-transported sodium ion. Using computational electrophysiology, we simulate the electrochemical gradient necessary for F- export in Fluc and reveal details about the coordination and hydration of both F- and the central sodium ion. The procedures described here delineate the specifics of these advanced techniques and can also be adapted to investigate other membrane protein systems.

Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Uncovering the Mechanism of the Proton-Coupled Fluoride Transport in the CLCF Antiporter.

Fluoride is a natural antibiotic abundantly present in the environment and, in micromolar concentrations, is able to inhibit enzymes necessary for bacteria to survive. However, as is the case with many antibiotics, bacteria have evolved resistance methods, including through the use of recently discovered membrane proteins. One such protein is the CLCF F-/H+ antiporter protein, a member of the CLC superfamily of anion-transport proteins. Though previous studies have examined this F- transporter, many questions are still left unanswered. To reveal details of the transport mechanism used by CLCF, we have employed molecular dynamics simulations and umbrella sampling calculations. Our results have led to several discoveries, including the mechanism of proton import and how it is able to aid in the fluoride export. Additionally, we have determined the role of the previously identified residues Glu118, Glu318, Met79, and Tyr396. This work is among the first studies of the CLCF F-/H+ antiporter and is the first computational investigation to model the full transport process, proposing a mechanism which couples the F- export with the H+ import.

Histone tail electrostatics modulate E2-E3 enzyme dynamics: a gateway to regulate ubiquitination machinery.

BRCA1 (Breast Cancer-Associated Protein 1) is a human tumor suppressor that functions as an ubiquitin (Ub) ligase enzyme (E3) and plays a key role in genomic stability and DNA repair. Heterodimerization of BRCA1 with BARD1 (BRCA1-associated RING domain protein 1) is known to increase its Ub ligase activity and is important for its stability, and cooperative activation of UbcH5c (Ub conjugating enzyme (E2)). Recent studies demonstrate the importance of ubiquitination of the nucleosomal H2A C-terminal tail by BRCA1/BARD1-UbcH5c in which its mutations inhibit ubiquitination, predispose cells to chromosomal instability and greatly increase the likelihood of breast and ovarian cancer development. Due to the lack of molecular-level insight on the flexible and dis-ordered H2A C-tail, its ubiquitination mechanism by BRCA1/BARD1-UbcH5c and its function and relationship to cancer susceptibility remain elusive. Here, we use molecular dynamics simulations to provide molecular-level insights into the dynamics of the less-studied H2A C-tail and BRCA1/BARD1-UbcH5c on the nucleosome surface and their effect on ubiquitination. Our results precisely identify the key interactions and residues that trigger conformational transitions of BRCA1/BARD1-UbcH5c, and characterize the important role of histone electrostatics in their dynamics. We provide a mechanistic basis for the H2A C-tail lysine approach to UbcH5c and show the role of H2A C-tail and UbcH5c dynamics in lysine ubiquitination. Furthermore, our data demonstrate the potential for ubiquitination based on the lysine position of the C-tail. Altogether, the findings of this study provide unrevealed insights into the mechanism of H2A C-tail ubiquitination and help us understand the communication between Ub ligase/Ub conjugating enzymes (E3/E2) and nucleosome to regulate ubiquitination machinery, paving the way for the development of effective treatments for cancer and chronic pain.

The Impact of Inflammation-Induced Tumor Plasticity during Myeloid Transformation.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of mutated preleukemic cells. Individuals with CH are at an increased risk of developing hematopoietic malignancies. Here, we describe a novel animal model carrying a recurrent TET2 missense mutation frequently found in patients with CH and leukemia. In a fashion similar to CH, animals show signs of disease late in life when they develop a wide range of myeloid neoplasms, including acute myeloid leukemia (AML). Using single-cell transcriptomic profiling of the bone marrow, we show that disease progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population. The gene signature characteristic of this inflammatory population is associated with poor prognosis in patients with AML. Our study illustrates an example of collaboration between a genetic lesion found in CH and inflammation, leading to transformation and the establishment of blood neoplasms. SIGNIFICANCE: Progression from a preleukemic state to transformation, in the presence of TET2 mutations, is coupled with the emergence of inflammation and a novel population of inflammatory monocytes. Genes characteristic of this inflammatory population are associated with the worst prognosis in patients with AML. These studies connect inflammation to progression to leukemia. See related commentary by Pietras and DeGregori, p. 2234 . This article is highlighted in the In This Issue feature, p. 2221.

Biophysical and in silico characterization of NrtA: a protein-based host for aqueous nitrate and nitrite recognition.

Nitrate and nitrite are key components of the global nitrogen cycle. As such, Nature has evolved proteins as biological supramolecular hosts for the recognition, translocation, and transformation of both nitrate and nitrite. To understand the supramolecular principles that govern these anion-protein interactions, here, we employ a hybrid biophysical and in silico approach to characterize the thermodynamic properties and protein dynamics of NrtA from the cyanobacterium Synechocystis sp. PCC 6803 for the recognition of nitrate and nitrite.

M2 amphipathic helices facilitate pH-dependent conformational transition in influenza A virus.

The matrix-2 (M2) protein from influenza A virus is a tetrameric, integral transmembrane (TM) protein that plays a vital role in viral replication by proton flux into the virus. The His37 tetrad is a pH sensor in the center of the M2 TM helix that activates the channel in response to the low endosomal pH. M2 consists of different regions that are believed to be involved in membrane targeting, packaging, nucleocapsid binding, and proton transport. Although M2 has been the target of many experimental and theoretical studies that have led to significant insights into its structure and function under differing conditions, the main mechanism of proton transport, its conformational dynamics, and the role of the amphipathic helices (AHs) on proton conductance remain elusive. To this end, we have applied explicit solvent constant pH molecular dynamics using the multisite λ-dynamics approach (CpHMDMSλD) to investigate the buried ionizable residues comprehensively and to elucidate their effect on the conformational transition. Our model recapitulates the pH-dependent conformational transition of M2 from closed to open state when the AH domain is included in the M2 construct, revealing the role of the amphipathic helices on this transition and shedding light on the proton-transport mechanism. This work demonstrates the importance of including the amphipathic helices in future experimental and theoretical studies of ion channels. Finally, our work shows that explicit solvent CpHMDMSλD provides a realistic pH-dependent model for membrane proteins.

Insight into wild-type and T1372E TET2-mediated 5hmC oxidation using ab initio QM/MM calculations.

Ten-eleven translocation 2 (TET2) is an Fe/α-ketoglutarate (α-KG) dependent enzyme that dealkylates 5-methylcytosine (5mC). The reaction mechanism involves a series of three sequential oxidations that convert 5mC to 5-hydroxy-methylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Our previous biochemical and computational studies uncovered an active site scaffold that is required for wild-type (WT) stepwise oxidation (Nat. Chem. Bio., 13, 181). We showed that the mutation of a single residue, T1372 to some amino acids, such as Glu, can impact the iterative oxidation steps and stop the oxidation of 5hmC to 5fC/caC. However, the source of the stalling at the first oxidation step by some mutant TET proteins still remains unclear. Here, we studied the catalytic mechanism of oxidation of 5hmC to 5fC by WT and T1372E TET2 using an ab initio quantum mechanical/molecular mechanical (QM/MM) approach. Our results suggest that the rate limiting step for WT TET2 involves a hydrogen atom abstraction from the hydroxyl group of 5hmC by the ferryl moiety in the WT. By contrast, our calculations for the T1372E mutant indicate that the rate limiting step for this variant corresponds to a second proton abstraction and the calculated barrier is almost twice as large as for WT TET2. Our results suggest that the large barrier for the 5hmC to 5fC oxidation in this mutant is due (at least in part) to the unfavorable orientation of the substrate in the active site. Combined electron localization function (ELF) and non-covalent interaction (NCI) analyses provide a qualitative description of the evolution of the electronic structure of the active site along the reaction path. Energy decomposition analysis (EDA) has been performed on the WT to investigate the impact of each MM residue on catalytic activity.

Tinker-HP: a massively parallel molecular dynamics package for multiscale simulations of large complex systems with advanced point dipole polarizable force fields.

We present Tinker-HP, a massively MPI parallel package dedicated to classical molecular dynamics (MD) and to multiscale simulations, using advanced polarizable force fields (PFF) encompassing distributed multipoles electrostatics. Tinker-HP is an evolution of the popular Tinker package code that conserves its simplicity of use and its reference double precision implementation for CPUs. Grounded on interdisciplinary efforts with applied mathematics, Tinker-HP allows for long polarizable MD simulations on large systems up to millions of atoms. We detail in the paper the newly developed extension of massively parallel 3D spatial decomposition to point dipole polarizable models as well as their coupling to efficient Krylov iterative and non-iterative polarization solvers. The design of the code allows the use of various computer systems ranging from laboratory workstations to modern petascale supercomputers with thousands of cores. Tinker-HP proposes therefore the first high-performance scalable CPU computing environment for the development of next generation point dipole PFFs and for production simulations. Strategies linking Tinker-HP to Quantum Mechanics (QM) in the framework of multiscale polarizable self-consistent QM/MD simulations are also provided. The possibilities, performances and scalability of the software are demonstrated via benchmarks calculations using the polarizable AMOEBA force field on systems ranging from large water boxes of increasing size and ionic liquids to (very) large biosystems encompassing several proteins as well as the complete satellite tobacco mosaic virus and ribosome structures. For small systems, Tinker-HP appears to be competitive with the Tinker-OpenMM GPU implementation of Tinker. As the system size grows, Tinker-HP remains operational thanks to its access to distributed memory and takes advantage of its new algorithmic enabling for stable long timescale polarizable simulations. Overall, a several thousand-fold acceleration over a single-core computation is observed for the largest systems. The extension of the present CPU implementation of Tinker-HP to other computational platforms is discussed.

Computational and experimental characterization of a pyrrolidinium-based ionic liquid for electrolyte applications.

The development of Li-ion batteries for energy storage has received significant attention. The synthesis and characterization of electrolytes in these batteries are an important component of this development. Ionic liquids (ILs) have been proposed as possible electrolytes in these devices. Thus, the accurate determination of thermophysical properties for these solvents becomes important for determining their applicability as electrolytes. In this contribution, we present the synthesis and experimental/computational characterization of thermodynamic and transport properties of a pyrrolidinium based ionic liquid as a first step to investigate the possible applicability of this class of ILs for Li-ion batteries. A quantum mechanical-based force field with many-body polarizable interactions has been developed for the simulation of spirocyclic pyrrolidinium, [sPyr+], with BF4- and Li+. Molecular dynamics calculations employing intra-molecular polarization predicted larger heat of vaporization and self-diffusion coefficients and smaller densities in comparison with the model without intra-molecular polarization, indicating that the inclusion of this term can significantly effect the inter-ionic interactions. The calculated properties are in good agreement with available experimental data for similar IL pairs and isothermal titration calorimetry data for [sPyr+][BF4-].

Computational investigation of O2 diffusion through an intra-molecular tunnel in AlkB; influence of polarization on O2 transport.

E. Coli AlkB catalyzes the direct dealkylation of various alkylated bases in damaged DNA. The diffusion of molecular oxygen to the active site in AlkB is an essential step for the oxidative dealkylation activity. Despite detailed studies on the stepwise oxidation mechanism of AlkB, there is no conclusive picture of how O2 molecules reach the active site of the protein. Yu et al. (Nature, 439, 879) proposed the existence of an intra-molecular tunnel based on their initial crystal structures of AlkB. We have employed computational simulations to investigate possible migration pathways inside AlkB for O2 molecules. Extensive molecular dynamics (MD) simulations, including explicit ligand sampling and potential of mean force (PMF) calculations, have been performed to provide a microscopic description of the O2 delivery pathway in AlkB. Analysis of intra-molecular tunnels using the CAVER software indicates two possible pathways for O2 to diffuse into the AlkB active site. Explicit ligand sampling simulations suggests that only one of these tunnels provides a viable route. The free energy path for an oxygen molecule to travel along each of these tunnels has been determined with AMBER and AMOEBA. Both PMFs indicate passive transport of O2 from the surface of the protein. However, the inclusion of explicit polarization shows a very large barrier for diffusion of the co-substrate out of the active site, compared with the non-polarizable potential. In addition, our results suggest that the mutation of a conserved residue along the tunnel, Y178, has dramatic effects on the dynamics of AlkB and on the transport of O2 along the tunnel.

Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine.

Ten-eleven translocation (TET) enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) to yield 5-hydroxymethylcytosine (hmC) and the rarer bases 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Stepwise oxidation obscures how each individual base forms and functions in epigenetic regulation, and prompts the question of whether TET enzymes primarily serve to generate hmC or are adapted to produce fC and caC as well. By mutating a single, conserved active site residue in human TET2, Thr1372, we uncovered enzyme variants that permit oxidation to hmC but largely eliminate fC and caC. Biochemical analyses, combined with molecular dynamics simulations, elucidated an active site scaffold that is required for wild-type (WT) stepwise oxidation and that, when perturbed, explains the mutants' hmC-stalling phenotype. Our results suggest that the TET2 active site is shaped to enable higher-order oxidation and provide the first TET variants that could be used to probe the biological functions of hmC separately from fC and caC.

Modeling Molecular Interactions in Water: From Pairwise to Many-Body Potential Energy Functions.

Almost 50 years have passed from the first computer simulations of water, and a large number of molecular models have been proposed since then to elucidate the unique behavior of water across different phases. In this article, we review the recent progress in the development of analytical potential energy functions that aim at correctly representing many-body effects. Starting from the many-body expansion of the interaction energy, specific focus is on different classes of potential energy functions built upon a hierarchy of approximations and on their ability to accurately reproduce reference data obtained from state-of-the-art electronic structure calculations and experimental measurements. We show that most recent potential energy functions, which include explicit short-range representations of two-body and three-body effects along with a physically correct description of many-body effects at all distances, predict the properties of water from the gas to the condensed phase with unprecedented accuracy, thus opening the door to the long-sought "universal model" capable of describing the behavior of water under different conditions and in different environments.

Development of AMOEBA force field for 1,3-dimethylimidazolium based ionic liquids.

The development of AMOEBA (a multipolar polarizable force field) for imidazolium based ionic liquids is presented. Our parametrization method follows the AMOEBA procedure and introduces the use of QM intermolecular total interactions as well as QM energy decomposition analysis (EDA) to fit individual interaction energy components. The distributed multipoles for the cation and anions have been derived using both the Gaussian distributed multipole analysis (GDMA) and Gaussian electrostatic model-distributed multipole (GEM-DM) methods.1 The intermolecular interactions of a 1,3-dimethylimidazolium [dmim(+)] cation with various anions, including fluoride [F(-)], chloride [Cl(-)], nitrate [NO(3)(-)], and tetraflorouborate [BF(4)(-)], were studied using quantum chemistry calculations at the MP2/6-311G(d,p) level of theory. Energy decomposition analysis was performed for each pair using the restricted variational space decomposition approach (RVS) at the HF/6-311G(d,p) level. The new force field was validated by running a series of molecular dynamic (MD) simulations and by analyzing thermodynamic and structural properties of these systems. A number of thermodynamic properties obtained from MD simulations were compared with available experimental data. The ionic liquid structure reproduced using the AMOEBA force field is also compared with the data from neutron diffraction experiment and other MD simulations. Employing GEM-DM force fields resulted in a good agreement on liquid densities ρ, enthalpies of vaporization ΔH(vap), and diffusion coefficients D(±) in comparison with conventional force fields.

Mechanisms and kinetics of thiotepa and tepa hydrolysis: DFT study.

N,N',N″-triethylenethiophosphoramide (Thiotepa) and its oxo analogue (Tepa) as the major metabolite are trifunctional alkylating agents with a broad spectrum of antitumor activity. In vivo and vitro studies show alkylation of DNA by Thiotepa and Tepa can follow two pathways, but it remains unclear which pathway represents the precise mechanism of action. In pathway 1, these agents are capable of forming cross-links with DNA molecules via two different mechanisms. In the first mechanism, the ring opening reaction is initiated by protonating the aziridine, which then becomes the primary target of nucleophilic attack by the N7-Guanine. The second one is a direct nucleophilic ring opening of aziridyl group. Thiotepa and Tepa in pathway 2, act as a cell penetrating carrier for aziridine, which is released via hydrolysis. The released aziridine can form a cross-link with N7-Guanine. In this study, we calculated the activation free energy and kinetic rate constant for hydrolysis of these agents and explored interaction of aziridine with Guanine to predict the most probable mechanism by applying density functional theory (DFT) using B3LYP method. In addition, solvent effect was introduced using the conductor-like polarizable continuum model (CPCM) in water, THF and diethylether. Hyperconjugation stabilization factors that have an effect on stability of generated transition state were investigated by natural bond order (NBO) analysis. Furthermore, quantum theory of atoms in molecules (QTAIM) analysis was performed to extract the bond critical points (BCP) properties, because the electron densities can be considered as a good description of the strength of different types of interactions.