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.

Light Chain Mutation Effects on the Dynamics of Thrombin.

Thrombin plays an important role in the process of hemostasis and blood coagulation. Studies in thrombin can help us find ways to treat cancer because thrombin is able to reduce the characteristic hypercoagulability of cancer. Thrombin is composed of two chains, the light chain and the heavy chain. The function of the heavy chain has been largely explored, while the function of the light chain was obscured until several disease-associated mutations in the light chain come to light. In this study, we want to explore the dynamic and conformation effects of mutations on the light chain further to determine possible associations between mutation, conformational changes, and disease. The study, which is a follow-up for our studies on apo thrombin and the mutant, ΔK9, mainly focuses on the mutants E8K and R4A. E8K is a disease-associated mutation, and R4A is used to study the role of Arg4, which is suggested experimentally to play a critical role for thrombin's catalytic activities. We performed five all-atom one microsecond-scale molecular dynamics (MD) simulations for both E8K and R4A, and quantified the changes in the conformational ensemble of the mutants. From the root-mean-square fluctuations (RMSF) for the α-carbons, we find that the atomic fluctuations change in the mutants in the 60s loop and γ loop. The correlation coefficients for the α-carbons indicate that the correlation relation for atom-pairs in the protein is also impacted. The clustering analysis and the principal component analysis (PCA) consistently tell us that the catalytic pocket and the regulatory loops are destabilized by the mutations. We also find that there are two binding modes for Na+ by clustering the vector difference between the Na+ ions and the 220s loop. After further analysis, we find that there is a relation between the Na+ binding and the rigidification of the γ loop, which may shed light on the mysterious role of the γ loop in thrombin.

Na+-binding modes involved in thrombin's allosteric response as revealed by molecular dynamics simulations, correlation networks and Markov modeling.

The monovalent sodium ion (Na+) is a critical modulator of thrombin. However, the mechanism of thrombin's activation by Na+ has been widely debated for more than twenty years. Details of the linkage between thrombin and Na+ remain vague due to limited temporal and spatial resolution in experiments. In this work, we combine microsecond scale atomic-detailed molecular dynamics simulations with correlation network analyses and hidden Markov modeling to probe the detailed thermodynamic and kinetic picture of Na+-binding events and their resulting allosteric responses in thrombin. We reveal that ASP189 and ALA190 comprise a stable Na+-binding site (referred as "inner" Na+-binding site) along with the previously known one (referred as "outer" Na+-binding site). The corresponding newly identified Na+-binding mode introduces significant allosteric responses in thrombin's regulatory regions by stabilizing selected torsion angles of residues responsive to Na+-binding. Our Markov model indicates that the bound Na+ prefers to transfer between the two Na+-binding sites when an unbinding event takes place. These results suggest a testable hypothesis of a substrate-driven Na+ migration (ΔG ∼ 1.7 kcal mol-1) from the "inner" Na+-binding site to the "outer" one during thrombin's catalytic activities. The binding of a Na+ ion at the "inner" Na+-binding site should be inferred as a prerequisite for thrombin's efficient recognition to the substrate, which opens a new angle for our understanding of Na+-binding's allosteric activation on thrombin and sheds light on detailed processes in thrombin's activation.

Probing light chain mutation effects on thrombin via molecular dynamics simulations and machine learning.

Thrombin is a key component for chemotherapeutic and antithrombotic therapy development. As the physiologic and pathologic roles of the light chain still remain vague, here, we continue previous efforts to understand the impacts of the disease-associated single deletion of LYS9 in the light chain. By combining supervised and unsupervised machine learning methodologies and more traditional structural analyses on data from 10 µs molecular dynamics simulations, we show that the conformational ensemble of the ΔK9 mutant is significantly perturbed. Our analyses consistently indicate that LYS9 deletion destabilizes both the catalytic cleft and regulatory functional regions and result in some conformational changes that occur in tens to hundreds of nanosecond scaled motions. We also reveal that the two forms of thrombin each prefer a distinct binding mode of a Na+ ion. We expand our understanding of previous experimental observations and shed light on the mechanisms of the LYS9 deletion associated bleeding disorder by providing consistent but more quantitative and detailed structural analyses than early studies in literature. With a novel application of supervised learning, i.e. the decision tree learning on the hydrogen bonding features in the wild-type and ΔK9 mutant forms of thrombin, we predict that seven pairs of critical hydrogen bonding interactions are significant for establishing distinct behaviors of wild-type thrombin and its ΔK9 mutant form. Our calculations indicate the LYS9 in the light chain has both localized and long-range allosteric effects on thrombin, supporting the opinion that light chain has an important role as an allosteric effector.

Using correlated motions to determine sufficient sampling times for molecular dynamics.

Here we present a time-dependent correlation method that provides insight into how long a system takes to grow into its equal-time (Pearson) correlation. We also show a usage of an extant time-lagged correlation method that indicates the time for parts of a system to become decorrelated, relative to equal-time correlation. Given a completed simulation (or set of simulations), these tools estimate (i) how long of a simulation of the same system would be sufficient to observe the same correlated motions, (ii) if patterns of observed correlated motions indicate events beyond the timescale of the simulation, and (iii) how long of a simulation is needed to observe these longer timescale events. We view this method as a decision-support tool that will aid researchers in determining necessary sampling times. In principle, this tool is extendable to any multidimensional time series data with a notion of correlated fluctuations; however, here we limit our discussion to data from molecular-dynamics simulations.

Visualizing correlated motion with HDBSCAN clustering.

Correlated motion analysis provides a method for understanding communication between and dynamic similarities of biopolymer residues and domains. The typical equal-time correlation matrices-frequently visualized with pseudo-colorings or heat maps-quickly convey large regions of highly correlated motion but hide more subtle similarities of motion. Here we propose a complementary method for visualizing correlations within proteins (or general biopolymers) that quickly conveys intuition about which residues have a similar dynamic behavior. For grouping residues, we use the recently developed non-parametric clustering algorithm HDBSCAN. Although the method we propose here can be used to group residues using correlation as a similarity matrix-the most straightforward and intuitive method-it can also be used to more generally determine groups of residues which have similar dynamic properties. We term these latter groups "Dynamic Domains", as they are based not on spatial closeness but rather closeness in the column space of a correlation matrix. We provide examples of this method across three human proteins of varying size and function-the Nf-Kappa-Beta essential modulator, the clotting promoter Thrombin and the mismatch repair protein (dimer) complex MutS-alpha. Although the examples presented here are from all-atom molecular dynamics simulations, this visualization technique can also be used on correlations matrices built from any ensembles of conformations from experiment or computation.

Spillover of Newcastle disease viruses from poultry to wild birds in Guangdong province, southern China.

Despite intensive vaccination programs in many countries, including China, Newcastle disease has been reported sporadically and is still a significant threat to the poultry industry in China. Newcastle disease virus (NDV) is infectious for at least 250 bird species, but the role of wild birds in virus epidemiology remains largely unknown. Fourteen NDV isolates were obtained from 2040 samples collected from wild birds or the environment in Guangdong province, southern China, from 2013 to 2015. The isolation rate was the highest in the period of wintering and lowest during the periods of spring migration, nesting, and postnesting. A maximum clade credibility phylogenetic analysis revealed that at least four genotypes circulate in southern China: three class II genotypes (II, VI, and IX) and one class I (1b). We also demonstrated that most isolates from wild birds were highly similar to isolates from poultry, and two isolates were linked to viruses from wild birds in northern China. These data suggested that wild birds could disseminate NDV and poultry-derived viruses may spillover to wild birds. Accordingly, vaccine development and poultry management strategies should be considered to prevent future NDV outbreaks, particularly given the strength of the poultry industry in developing countries, such as China.

Mechanistic insights into thrombin's switch between "slow" and "fast" forms.

Thrombin is a multifunctional enzyme that plays an important role in blood coagulation, cell growth, and metastasis. Depending upon the binding of sodium ions, thrombin presents significantly different enzymatic activities. In the environment with sodium ions, thrombin is highly active in cleaving the coagulated substrates and this is referred to as the "fast" form; in the environment without sodium ions, thrombin turns catalytically less active and is in the "slow" form. Although many experimental studies over the last two decades have attempted to reveal the structural and kinetic differences between these two forms, it remains vague and disputed how the functional switch between the "fast" and "slow" forms is mediated by Na+ cations. In this work, we employ microsecond-scale all-atom molecular dynamics simulations to investigate the differences in the structural ensembles in sodium-bound/unbound and potassium-bound/unbound thrombin. Our calculations indicate that the regulatory regions, including the 60s, γ loops, and exosite I and II, are primarily affected by both the bound and unbound cations. Conformational free energy surfaces, estimated from principal component analysis, further reveal the existence of multiple conformational states. The binding of a cation introduces changes in the distribution of these states. Through comparisons with potassium-binding, the binding of sodium ions appears to shift the population toward conformational states that might be catalytically favorable. Our study of thrombin in the presence of sodium/potassium ions suggests Na+-mediated generalized allostery is the mechanism of thrombin's functional switch between the "fast" and "slow" forms.

Immune Responses of Chickens Infected with Wild Bird-Origin H5N6 Avian Influenza Virus.

Since April 2014, new infections of H5N6 avian influenza virus (AIV) in humans and domestic poultry have caused considerable economic losses in the poultry industry and posed an enormous threat to human health worldwide. In previous research using gene sequence and phylogenetic analysis, we reported that H5N6 AIV isolated in February 2015 (ZH283) in Pallas's sandgrouse was highly similar to that isolated in a human in December 2015 (A/Guangdong/ZQ874/2015), whereas a virus (i.e., SW8) isolated in oriental magpie-robin in 2014 was highly similar to that of A/chicken/Dongguan/2690/2013 (H5N6). However, the pathogenicity, transmissibility, and host immune-related response of chickens infected by those wild bird-origin H5N6 AIVs remain unknown. In response, we examined the viral distribution and mRNA expression profiles of immune-related genes in chickens infected with both viruses. Results showed that the H5N6 AIVs were highly pathogenic to chickens and caused not only systemic infection in multiple tissues, but also 100% mortality within 3-5 days post-infection. Additionally, ZH283 efficiently replicated in all tested tissues and transmitted among chickens more rapidly than SW8. Moreover, quantitative real-time polymerase chain reaction analysis showed that following infection with H5N6, AIVs immune-related genes remained active in a tissue-dependent manner, as well as that ZH283 induced mRNA expression profiles such as TLR3, TLR7, IL-6, TNF-α, IL-1ß, IL-10, IL-8, and MHC-II to a greater extent than SW8 in the tested tissues of infected chickens. Altogether, our findings help to illuminate the pathogenesis and immunologic mechanisms of H5N6 AIVs in chickens.

Molecular dynamics simulations of aptamer-binding reveal generalized allostery in thrombin.

Thrombin is an attractive target for antithrombotic therapy due to its central role in thrombosis and hemostasis as well as its role in inducing tumor growth, metastasis, and tumor invasion. The thrombin-binding DNA aptamer (TBA), is under investigation for anticoagulant drugs. Although aptamer binding experiments have been revealed various effects on thrombin's enzymatic activities, the detailed picture of the thrombin's allostery from TBA binding is still unclear. To investigate thrombin's response to the aptamer-binding at the molecular level, we compare the mechanical properties and free energy landscapes of the free and aptamer-bound thrombin using microsecond-scale all-atom GPU-based molecular dynamics simulations. Our calculations on residue fluctuations and coupling illustrate the allosteric effects of aptamer-binding at the atomic level, highlighting the exosite II, 60s, γ and the sodium loops, and the alpha helix region in the light chains involved in the allosteric changes. This level of details clarifies the mechanisms of previous experimentally demonstrated phenomena, and provides a prediction of the reduced autolysis rate after aptamer-binding. The shifts in thrombin's ensemble of conformations and free energy surfaces after aptamer-binding demonstrate that the presence of bound-aptamer restricts the conformational freedom of thrombin suggesting that conformational selection, i.e. generalized allostery, is the dominant mechanism of thrombin-aptamer binding. The profound perturbation on thrombin's mechanical and thermodynamic properties due to the aptamer-binding, which was revealed comprehensively as a generalized allostery in this work, may be exploited in further drug discovery and development.

Uncovering Large-Scale Conformational Change in Molecular Dynamics without Prior Knowledge.

As the length of molecular dynamics (MD) trajectories grows with increasing computational power, so does the importance of clustering methods for partitioning trajectories into conformational bins. Of the methods available, the vast majority require users to either have some a priori knowledge about the system to be clustered or to tune clustering parameters through trial and error. Here we present non-parametric uses of two modern clustering techniques suitable for first-pass investigation of an MD trajectory. Being non-parametric, these methods require neither prior knowledge nor parameter tuning. The first method, HDBSCAN, is fast-relative to other popular clustering methods-and is able to group unstructured or intrinsically disordered systems (such as intrinsically disordered proteins, or IDPs) into bins that represent global conformational shifts. HDBSCAN is also useful for determining the overall stability of a system-as it tends to group stable systems into one or two bins-and identifying transition events between metastable states. The second method, iMWK-Means, with explicit rescaling followed by K-Means, while slower than HDBSCAN, performs well with stable, structured systems such as folded proteins and is able to identify higher resolution details such as changes in relative position of secondary structural elements. Used in conjunction, these clustering methods allow a user to discern quickly and without prior knowledge the stability of a simulated system and identify both local and global conformational changes.

Combining capillary electrophoresis and next-generation sequencing for aptamer selection.

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or "aptamers" because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4% aptamer content before selection to >15% aptamer content.

Spirometra (Pseudophyllidea, Diphyllobothriidae) severely infecting wild-caught snakes from food markets in Guangzhou and Shenzhen, Guangdong, China: implications for public health.

Sparganosis is a zoonotic disease caused by the spargana of Spirometra, and snake is one of the important intermediate hosts of spargana. In some areas of China, snake is regarded as popular delicious food, and such a food habit potentially increases the prevalence of human sparganosis. To understand the prevalence of Spirometra in snakes in food markets, we conducted a study in two representative cities (Guangzhou and Shenzhen), during January-August 2013. A total of 456 snakes of 13 species were examined and 251 individuals of 10 species were infected by Spirometra, accounting for 55.0% of the total samples. The worm burden per infected snake ranged from 1 to 213, and the prevalence in the 13 species was 0∼96.2%. More than half (58.1%) of the spargana were located in muscular tissue, 25.6% in subcutaneous tissue, and 16.3% in coelomic cavity. The results indicated that Spirometra severely infected snakes in food markets in Guangzhou and Shenzhen, implying that eating snakes has great health risk and improper cooking methods may increase the risk of Spirometra infection in humans in China. Additional steps should be considered by the governments and public health agencies to prevent the risk of snake-associated Spirometra infections in humans.