Temperature Replica Exchange Molecular Dynamics Research Articles

Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a β-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the β-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual β-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

A theoretical study on the activity and selectivity of IDO/TDO inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO) is a tryptophan (Trp) metabolic enzyme along the kynurenine (NFK) pathway. Under pathological conditions, IDO overexpressed by tumor cells causes depletion of tryptophan and the accumulation of metabolic products, which inhibit the local immune response and form immune escape. Therefore, the suppression of IDO activity is one of the strategies for tumor immunotherapy, and drug design for this target has been the focus of research for more than two decades. Apart from IDO, tryptophan dioxygenase (TDO) of the same family can also catalyze the same biochemical reaction in the human body, but it has different tissue distribution and substrate selectivity from IDO. Based on the principle of drug design with high potency and low cross-reactivity to specific targets, in this subject, the activity and selectivity of IDO and TDO toward small molecular inhibitors were studied from the perspective of thermodynamics and kinetics. The aim was to elucidate the structural requirements for achieving favorable biological activity and selectivity of IDO and TDO inhibitors. Specifically, the interactions of inhibitors from eight families with IDO and TDO were initially investigated through molecular docking and molecular dynamics simulations, and the thermodynamic data for binding of inhibitors were predicted by the molecular mechanics/generalized Born surface area (MM/GBSA) method. Secondly, we explored the free energy landscape of JKloops, the kinetic control element of IDO/TDO, using temperature replica exchange molecular dynamics (T-REMD) simulations and elucidated the connection between the rules of IDO/TDO conformational changes and the inhibitor selectivity mechanism. Furthermore, the binding and dissociation processes of the C1 inhibitor (NLG919) were simulated by the adaptive steering molecular dynamics (ASMD) method, which not only addressed the possible stable, metastable, and transition states for C1 inhibitor-IDO/TDO interactions, but also accurately predicted kinetic data for C1 inhibitor binding and dissociation. In conclusion, we have constructed a complete process from enzyme (IDO/TDO) conformational activation to inhibitor binding/dissociation and used the thermodynamic and kinetic data of each link as clues to verify the control mechanism of IDO/TDO on inhibitor selectivity. This is of great significance for us to understand the design principles of tumor immunotherapy drugs and to avoid drug resistance of immunotherapy drugs.

Silver nanoparticles alter the dimerization of Aβ42 studied by REMD simulations.

The aggregation of amyloid beta (Aβ) peptides is associated with the development of Alzheimer's disease (AD). However, there has been a growing belief that the oligomerization of Aβ species in different environments has a neurotoxic effect on the patient's brain, causing damage. It is necessary to comprehend the compositions of Aβ oligomers in order to develop medications that may effectively inhibit these neurotoxic forms that affect the nervous system of AD patients. Thus, dissociation or inhibition of Aβ aggregation may be able to prevent AD. To date, the search for traditional agents and biomolecules has largely been unsuccessful. In this context, nanoparticles have emerged as potential candidates to directly inhibit the formation of Aβ oligomers. The oligomerization of the dimeric Aβ peptides with or without the influence of a silver nanoparticle was thus investigated using temperature replica-exchange molecular dynamics (REMD) simulations. The physical insights into the dimeric Aβ oligomerization were clarified by analyzing intermolecular contact maps, the free energy landscape of the dimeric oligomer, secondary structure terms, etc. The difference in obtained metrics between Aβ with or without a silver nanoparticle provides a picture of the influence of silver nanoparticles on the oligomerization process. The underlying mechanisms that are involved in altering Aβ oligomerization will be discussed. The obtained results may play an important role in searching for Aβ inhibitor pathways.

Characterizing the transmembrane domains of ADAM10 and BACE1 and the impact of membrane composition

The β-secretase, BACE1, and the α-secretase, ADAM10, are known to competitively cleave amyloid precursor protein (APP) in the amyloid cascades of Alzheimer’s disease. Cleavage of APP by BACE1 produces a 99-residue C-terminal peptide (APP-C99) that is subsequently cleaved by γ-secretase to form amyloid-β (Aβ) protein, whereas cleavage of APP by ADAM10 is nonamyloidogenic. It has been speculated that ADAM10/APP and BACE1/APP interactions are regulated by colocalization within and outside of liquid-ordered membrane domains; however, the mechanism of this regulation and the character of the proteins’ transmembrane domains are not well understood. In this work, we have developed and characterized minimal congener sequences for the transmembrane domains of ADAM10 and BACE1 using a multiscale modeling approach combining both temperature replica exchange and conventional molecular dynamics simulations based on the coarse-grained Martini2.2 and all-atom CHARMM36 force fields. Our results show that membrane composition impacts the character of the transmembrane domains of BACE1 and ADAM10, adding credence to the speculation that membrane domains are involved in the etiology of Alzheimer’s disease.

Structural Space of the Duffy Antigen/Receptor for Chemokines' Intrinsically Disordered Ectodomain 1 Explored by Temperature Replica-Exchange Molecular Dynamics Simulations.

Plasmodium vivax malaria affects 14 million people each year. Its invasion requires interactions between the parasitic Duffy-binding protein (PvDBP) and the N-terminal extracellular domain (ECD1) of the host's Duffy antigen/receptor for chemokines (DARC). ECD1 is highly flexible and intrinsically disordered, therefore it can adopt different conformations. We computationally modeled the challenging ECD1 local structure. With T-REMD simulations, we sampled its dynamic behavior and collected its most representative conformations. Our results suggest that most of the DARC ECD1 domain remains in a disordered state during the simulated time. Globular local conformations are found in the analyzed local free-energy minima. These globular conformations share an α-helix spanning residues Ser18 to Ser29 and in many cases they comprise an antiparallel β-sheet, whose β-strands are formed around residues Leu10 and Ala49. The formation of a parallel β-sheet is almost negligible. So far, progress in understanding the mechanisms forming the basis of the P. vivax malaria infection of reticulocytes has been hampered by experimental difficulties, along with a lack of DARC structural information. Our collection of the most probable ECD1 structural conformations will help to advance modeling of the DARC structure and to explore DARC-ECD1 interactions with a range of physiological and pathological ligands.

Morphology of a Transmembrane Aβ42 Tetramer via REMD Simulations.

The folding/misfolding of membrane-permiable Amyloid beta (Aβ) peptides is likely associated with the advancing stage of Alzheimer's disease (AD) by disrupting Ca2+ homeostasis. In this context, the aggregation of four transmembrane Aβ17-42 peptides was investigated using temperature replica-exchange molecular dynamics (REMD) simulations. The obtained results indicated that the secondary structure of transmembrane Aβ peptides tends to have different propensities compared to those in solution. Interestingly, the residues favorably forming β-structure were interleaved by residues rigidly adopting turn-structure. A combination of β and turn regions likely forms a pore structure. Six morphologies of 4Aβ were found over the free energy landscape and clustering analyses. Among these, the morphologies include (1) Aβ binding onto the membrane surface and three transmembrane Aβ; (2) three helical and coil transmembrane Aβ; (3) four helical transmembrane Aβ; (4) three helical and one β-hairpin transmembrane Aβ; (5) two helical and two β-strand transmembrane Aβ; and (6) three β-strand and one helical transmembrane Aβ. Although the formation of the β-barrel structure was not observed during the 0.28 ms─long MD simulation, the structure is likely to form when the simulation time is further extended.

Conformational dynamics of RNA G4C2 and G2C4 repeat expansions causing ALS/FTD using NMR and molecular dynamics studies.

G4C2 and G2C4 repeat expansions in chromosome 9 open reading frame 72 (C9orf72) are the most common cause of genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or c9ALS/FTD. The gene is bidirectionally transcribed, producing G4C2 repeats [r(G4C2)exp] and G2C4 repeats [r(G2C4)exp]. The c9ALS/FTD repeat expansions are highly structured, and structural studies showed that r(G4C2)exp predominantly folds into a hairpin with a periodic array of 1×1 G/G internal loops and a G-quadruplex. A small molecule probe revealed that r(G4C2)exp also adopts a hairpin structure with 2×2 GG/GG internal loops. We studied the conformational dynamics adopted by 2×2 GG/GG loops using temperature replica exchange molecular dynamics (T-REMD) and further characterized the structure and underlying dynamics using traditional 2D NMR techniques. These studies showed that the loop's closing base pairs influence both structure and dynamics, particularly the configuration adopted around the glycosidic bond. Interestingly, r(G2C4) repeats, which fold into an array of 2×2 CC/CC internal loops, are not as dynamic. Collectively, these studies emphasize the unique sensitivity of r(G4C2)exp to small changes in stacking interactions, which is not observed in r(G2C4)exp, providing important considerations for further principles in structure-based drug design.

Role of Metal Cofactor in Enhanced Thermal Stability of Azurin.

Metal cofactors are critical centers for different biochemical processes of metalloproteins, and often, this metal coordination renders additional structural stability. In this study, we explore the additional stability conferred by the copper ion on azurin by analyzing both the apo and holo forms using temperature replica exchange molecular dynamics (REMD) data. We find a 14 K decrease in denaturation temperature for apo (406 K) azurin relative to that of holo (420 K), indicating a copper ion-induced additional thermal stability for holo azurin. The unfolding of apo azurin begins with the melting of α-helix and β-sheet V, similar to that of holo form. β-Sheets IV, VII, and VIII are comparatively more stable than other β-strands and melt at higher temperatures. Similar to holo azurin, the strong hydrophobic interactions among the apolar residues in the protein core is the key factor that renders high stability to apo protein as well. We construct free energy surfaces at different temperatures to capture the major conformations along the unfolding basins of the protein. Using contact maps from different basins we show the changes in the interaction between different residues along the unfolding pathway. Furthermore, we compare the Cα root-mean-square fluctuations (Cα-RMSF) and B-factor of all residues of apo and holo forms to understand the flexibility of different regions. The concerted displacement of α-helix and β-sheets V and VI from the protein core is another distinction we observe for apo compared to the holo form, where β-sheet VI was relatively stable.

Mutations affect the conformational plasticity of the β2-α2 loop of human prion protein.

The prion protein (PrP) is responsible for several transmissible spongiform encephalopathies such as bovine spongiform encephalopathy and chronic wasting disease in ruminants. In humans, PrP causes Creutzfeldt-Jacob disease, Kuru, and other diseases. The neurotoxicity of PrP manifests upon misfolding and aggregation, and the unfolding of its C-terminal domain is thought to be a key step towards unfolding. PrPs from animals resistant to prion diseases show distinct point mutations in their sequence with respect to human PrP. In this work, we test the hypothesis that the β2-α2 loop in disease-resistant species is characterized by a lower conformational heterogeneity than in human. We test our hypothesis with temperature replica-exchange molecular dynamics and with metadynamics simulations. Our results show that single mutations can alter the equilibrium between the 310-helical turn conformation and several β-turn conformations by reshaping both intra-loop interactions as well as interactions between the loop and the α3-helix.

Conformational dynamics of the cytoplasmic cap during desensitization of the hP2X3R.

The human P2X3 receptor (P2X3R) is a trimeric ligand-gated ion channel activated by extracellular ATP. X-ray crystallography revealed atomic structures of its apo (closed, resting), ATP-bound (open, activated), and ATP-bound (closed, desensitized) states. An intracellular ‘cytoplasmic cap’ was found to stabilize the open pore in the ATP-bound state. However, the cytoplasmic cap was not resolved in the resting or desensitized structures, suggesting a model in which transient formation of the cytoplasmic cap is associated with the opening of the ion pore. To study the functional coupling between (un)folding of the cytoplasmic cap and opening/closing of the P2X3R ion pore, we performed extensive all-atom molecular dynamics (MD) simulations combined with voltage-clamp electrophysiology and mutagenesis. Temperature replica-exchange MD simulations of P2X3R revealed the cytoplasmic cap's unfolding pathway and consequent ion pore closure. Our results show that the N- and C-terminal layers that form the cytoplasmic cap in the open state do not fully unfold during desensitization. By contrast, the partial unfolding of the lower C-terminal layer is sufficient to initiate pore closure/desensitization. In contrast, the N-terminal layers remain folded and keep their spatial proximity to the membrane. Interestingly, not all C-terminal secondary structures unfold, and some structural organization persists, which may allow for rapid transitions between the apo, open, and resting states. The main trigger of pore closure/desensitization is the loss of contact between the C-terminal and the N-terminal part of the cap. We investigated point mutations, aimed at (de)stabilizing the cytoplasmic cap, using free-energy calculations and electrophysiological recordings. These demonstrate that the kinetics of P2X3R desensitization depends on the folding-free energy of the cytoplasmic cap. In summary, our work reveals an allosteric coupling mechanism between the cytoplasmic cap and gating of the transmembrane ion pore in P2X receptors.

CHARMM-GUI Enhanced Sampler for various collective variables and enhanced sampling methods.

Enhanced sampling methodologies modifying underlying Hamiltonians can be used for the systems with a rugged potential energy surface that makes it hard to observe convergence using conventional unbiased molecular dynamics (MD) simulations. We present CHARMM-GUI Enhanced Sampler, a web-based tool to prepare various enhanced sampling simulations inputs with user-selected collective variables (CVs). Enhanced Sampler provides inputs for the following nine methods: accelerated MD, Gaussian accelerated MD, conformational flooding, metadynamics, adaptive biasing force, steered MD, temperature replica exchange MD, replica exchange solute tempering 2, and replica exchange umbrella sampling for the method-implemented MD packages including AMBER, CHARMM, GENESIS, GROMACS, NAMD, and OpenMM. Users only need to select a group of atoms via intuitive web-implementation in order to define commonly used nine CVs of interest: center of mass based distance, angle, dihedral, root-mean-square-distance, radius of gyration, distance projected on axis, two types of angles projected on axis, and coordination numbers. The enhanced sampling methods are tested with several biological systems to illustrate their efficiency over conventional MD. Enhanced Sampler with carefully optimized system-dependent parameters will help users to get meaningful results from their enhanced sampling simulations.

Accurate Structure Prediction for Protein Loops Based on Molecular Dynamics Simulations with RSFF2C.

Protein loops, connecting the α-helices and β-strands, are involved in many important biological processes. However, due to their conformational flexibility, it is still challenging to accurately determine three-dimensional (3D) structures of long loops experimentally and computationally. Herein, we present a systematic study of the protein loop structure prediction via a total of ∼850 μs molecular dynamics (MD) simulations. For a set of 15 long (10-16 residues) and solvent-exposed loops, we first evaluated the performance of four state-of-the-art loop modeling algorithms, DaReUS-Loop, Sphinx, Rosetta-NGK, and MODELLER, on each loop, and none of them could accurately predict the structures for most loops. Then, temperature replica exchange molecular dynamics (REMD) simulations were conducted with three recent force fields, RSFF2C with TIP3P water model, CHARMM36m with CHARMM-modified TIP3P, and AMBER ff19SB with OPC. We found that our recently developed residue-specific force field RSFF2C performed the best and successfully predicted 12 out of 15 loops with a root-mean-square deviation (RMSD) < 1.5 Å. As an alternative with lower computational cost, normal MD simulations at high temperatures (380, 500, and 620 K) were investigated. Temperature-dependent performance was observed for each force field, and, for RSFF2C+TIP3P, we found that three independent 100-ns MD simulations at 500 K gave comparable results with REMD simulations. These results suggest that MD simulations, especially with enhanced sampling techniques such as replica exchange, with the RSFF2C force field could be useful for accurate loop structure prediction.

Deciphering nucleotide modification-induced structure and stability changes

ABSTRACT Nucleotide modification in RNA controls a bevy of biological processes, including RNA degradation, gene expression, and gene editing. In turn, misregulation of modified nucleotides is associated with a host of chronic diseases and disorders. However, the molecular mechanisms driving these processes remain poorly understood. To partially address this knowledge gap, we used alchemical and temperature replica exchange molecular dynamics (TREMD) simulations on an RNA duplex and an analogous hairpin to probe the structural effects of modified and/or mutant nucleotides. The simulations successfully predict the modification/mutation-induced relative free energy change for complementary duplex formation, and structural analyses highlight mechanisms driving stability changes. Furthermore, TREMD simulations for a hairpin-forming RNA with and without modification provide reliable estimations of the energy landscape. Illuminating the impact of methylated and/or mutated nucleotides on the structure-function relationship and the folding energy landscape, the simulations provide insights into modification-induced alterations to the folding mechanics of the hairpin. The results here may be biologically significant as hairpins are widespread structure motifs that play critical roles in gene expression and regulation. Specifically, the tetraloop of the probed hairpin is phylogenetically abundant, and the stem mirrors a miRNA seed region whose modification has been implicated in epilepsy pathogenesis.

Computational Modeling of DNA-Nanotube Hybrid Conjugates for Biosensor Applications

DNA-single walled carbon nanotubes (SWNT) conjugates are promising materials for optical sensing of biological molecular analytes. Here, we use molecular dynamics (MD) simulations and machine learning techniques to examine these materials by themselves and in the presence of biological analytes. First, we explore the relationship between structural conformation, binding affinity, and kinetic stability for short single-stranded oligonucleotides adsorbed on SWNTs, by using MD simulations, temperature replica exchange MD calculations, and kinetic stability experimental measurements.

Increasing the Sampling Efficiency of Protein Conformational Change by Combining a Modified Replica Exchange Molecular Dynamics and Normal Mode Analysis.

Understanding conformational change at an atomic level is significant when determining a protein functional mechanism. Replica exchange molecular dynamics (REMD) is a widely used enhanced sampling method to explore protein conformational space. However, REMD with an explicit solvent model requires huge computational resources, immensely limiting its application. In this study, a variation of parallel tempering metadynamics (PTMetaD) with the omission of solvent-solvent interactions in exchange attempts and the use of low-frequency modes calculated by normal-mode analysis (NMA) as collective variables (CVs), namely ossPTMetaD, is proposed with the aim to accelerate MD simulations simultaneously in temperature and geometrical spaces. For testing the performance of ossPTMetaD, five protein systems with diverse biological functions and motion patterns were selected, including large-scale domain motion (AdK), flap movement (HIV-1 protease and BACE1), and DFG-motif flip in kinases (p38α and c-Abl). The simulation results showed that ossPTMetaD requires much fewer numbers of replicas than temperature REMD (T-REMD) with a reduction of ∼70% to achieve a similar exchange ratio. Although it does not obey the detailed balance condition, ossPTMetaD provides consistent results with T-REMD and experimental data. The high accessibility of the large conformational change of protein systems by ossPTMetaD, especially in simulating the very challenging DFG-motif flip of protein kinases, demonstrated its high efficiency and robustness in the characterization of the large-scale protein conformational change pathway and associated free energy profile.

Exploring Protocols to Build Reservoirs to Accelerate Temperature Replica Exchange MD Simulations.

Temperature replica exchange molecular dynamics (REMD) is a widely used enhanced sampling method for accelerating biomolecular simulations. During the past 2 decades, several variants of REMD have been developed to further improve the rate of conformational sampling of REMD. One such variant, reservoir REMD (RREMD), was shown to improve the rate of conformational sampling by around 5-20×. Despite the significant increase in the sampling speed, RREMD methods have not been widely used because of the difficulties in building the reservoir and also because of the code not being available on the graphics processing units (GPUs). In this work, we ported the Amber RREMD code onto GPUs making it 20× faster than the central processing unit code. Then, we explored protocols for building Boltzmann-weighted reservoirs as well as non-Boltzmann reservoirs and tested how each choice affects the accuracy of the resulting RREMD simulations. We show that, using the recommended protocols outlined here, RREMD simulations can accurately reproduce Boltzmann-weighted ensembles obtained by much more expensive conventional temperature-based REMD simulations, with at least 15× faster convergence rates even for larger proteins (>50 amino acids) compared to conventional REMD.

Oversampling Free Energy Perturbation Simulation in Determination of the Ligand-Binding Free Energy.

Determination of the ligand-binding affinity is an extremely interesting problem. Normally, the free energy perturbation (FEP) method provides an appropriate result. However, it is of great interest to improve the accuracy and precision of this method. In this context, temperature replica exchange molecular dynamics implementation of the FEP computational approach, which we call replica exchange free energy perturbation (REP) was proposed. In particular, during REP simulations, the system can easily escape from being trapped in local minima by exchanging configurations with high temperatures, resulting in significant improvement in the accuracy and precision of protein-ligand binding affinity calculations. The distribution of the decoupling free energy was enlarged, and its mean values were decreased. This results in changes in the magnitude of the calculated binding free energies as well as in alteration in the binding mechanism. Moreover, the REP correlation coefficient with respect to experiment ( RREP = 0.85 ± 0.15) is significantly boosted in comparison with the FEP one ( RFEP = 0.64 ± 0.30). Furthermore, the root-mean-square error (RMSE) of REP is also smaller than FEP, RMSEREP = 4.28 ± 0.69 versus RMSEFEP = 5.80 ± 1.11 kcal/mol, respectively. © 2019 Wiley Periodicals, Inc.

Sequence specificity despite intrinsic disorder: How a disease-associated Val/Met polymorphism rearranges tertiary interactions in a long disordered protein.

The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) in precursor brain-derived neurotrophic factor (BDNF) is one of the earliest SNPs to be associated with neuropsychiatric disorders, and the underlying molecular mechanism is unknown. Here we report on over 250 μs of fully-atomistic, explicit solvent, temperature replica-exchange molecular dynamics (MD) simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence. The simulations were able to correctly reproduce the location of both local and non-local secondary structure changes due to the Val66Met mutation, when compared with NMR spectroscopy. We find that the change in local structure is mediated via entropic and sequence specific effects. We developed a hierarchical sequence-based framework for analysis and conceptualization, which first identifies “blobs” of 4-15 residues representing local globular regions or linkers. We use this framework within a novel test for enrichment of higher-order (tertiary) structure in disordered proteins; the size and shape of each blob is extracted from MD simulation of the real protein (RP), and used to parameterize a self-avoiding heterogenous polymer (SAHP). The SAHP version of the BDNF prodomain suggested a protein segmented into three regions, with a central long, highly disordered polyampholyte linker separating two globular regions. This effective segmentation was also observed in full simulations of the RP, but the Val66Met substitution significantly increased interactions across the linker, as well as the number of participating residues. The Val66Met substitution replaces β-bridging between V66 and V94 (on either side of the linker) with specific side-chain interactions between M66 and M95. The protein backbone in the vicinity of M95 is then free to form β-bridges with residues 31-41 near the N-terminus, which condenses the protein. A significant role for Met/Met interactions is consistent with previously-observed non-local effects of the Val66Met SNP, as well as established interactions between the Met66 sequence and a Met-rich receptor that initiates neuronal growth cone retraction.

Oxidation-induced destabilization of the fibrinogen αC-domain dimer investigated by molecular dynamics simulations.

Upon activation, fibrinogen is converted to insoluble fibrin, which assembles into long strings called protofibrils. These aggregate laterally to form a fibrin matrix that stabilizes a blood clot. Lateral aggregation of protofibrils is mediated by the αC domain, a partially structured fragment located in a disordered region of fibrinogen. Polymerization of αC domains links multiple fibrin molecules with each other enabling the formation of thick fibrin fibers and a fibrin matrix that is stable but can also be digested by enzymes. However, oxidizing agents produced during the inflammatory response have been shown to cause thinner fibrin fibers resulting in denser clots, which are harder to proteolyze and pose the risk of deep vein thrombosis and lung embolism. Oxidation of Met476 located within the αC domain is thought to hinder its ability to polymerize disrupting the lateral aggregation of protofibrils and leading to the observed thinner fibers. How αC domains assemble into polymers is still unclear and yet this knowledge would shed light on the mechanism through which oxidation weakens the lateral aggregation of protofibrils. This study used temperature replica exchange molecular dynamics simulations to investigate the αC-domain dimer and how this is affected by oxidation of Met476 . Analysis of the trajectories revealed that multiple stable binding modes were sampled between two αC domains while oxidation decreased the likelihood of dimer formation. Furthermore, the side chain of Met476 was observed to act as a docking spot for the binding and this function was impaired by its conversion to methionine sulfoxide.

Importance of the Force Field Choice in Capturing Functionally Relevant Dynamics in the von Willebrand Factor.

Whether recent updates and new releases of atomistic force fields can model the structural and dynamical properties of proteins containing both folded and partially disordered domains is still unclear. To address this fundamental question, we tested eight recently released force fields against our set of nuclear magnetic resonance (NMR) observables for a complex and medically relevant system, the major factor VIII binding region on the von Willebrand factor. This biomedically important region comprises both a folded and a partially structured domain. By using an enhanced sampling technique (temperature replica-exchange molecular dynamics simulations), we find that some force fields indeed rise to the challenge and capture the structural and dynamical features of the NMR ensemble and, therefore, are the appropriate choice for simulations of proteins with partially structured domains. What is more, we show that only such force fields can qualitatively capture the effects of a pathogenic mutation on the structural ensemble.