Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.     

The Gating Mechanism of the Human Aquaporin 5 Revealed by Molecular Dynamics Simulations

Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5) shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.     

Molecular Dynamics Simulations of Insulin: Elucidating the Conformational Changes that Enable Its Binding

A sequence of complex conformational changes is required for insulin to bind to the insulin receptor. Recent experimental evidence points to the B chain C-terminal (BC-CT) as the location of these changes in insulin. Here, we present molecular dynamics simulations of insulin that reveal new insights into the structural changes occurring in the BC-CT. We find three key results: 1) The opening of the BC-CT is inherently stochastic and progresses through an open and then a “wide-open” conformation—the wide-open conformation is essential for receptor binding, but occurs only rarely. 2) The BC-CT opens with a zipper-like mechanism, with a hinge at the Phe24 residue, and is maintained in the dominant closed/inactive state by hydrophobic interactions of the neighboring Tyr26, the critical residue where opening of the BC-CT (activation of insulin) is initiated. 3) The mutation Y26N is a potential candidate as a therapeutic insulin analogue. Overall, our results suggest that the binding of insulin to its receptor is a highly dynamic and stochastic process, where initial docking occurs in an open conformation and full binding is facilitated through interactions of insulin receptor residues with insulin in its wide-open conformation.     

Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.     

Energetic Changes Caused by Antigenic Module Insertion in a Virus-Like Particle Revealed by Experiment and Molecular Dynamics Simulations

The success of recombinant virus-like particles (VLPs) for human papillomavirus and hepatitis B demonstrates the potential of VLPs as safe and efficacious vaccines. With new modular designs emerging, the effects of antigen module insertion on the self-assembly and structural integrity of VLPs should be clarified so as to better enabling improved design. Previous work has revealed insights into the molecular energetics of a VLP subunit, capsomere, comparing energetics within various solution conditions known to drive or inhibit self-assembly. In the present study, molecular dynamics (MD) simulations coupled with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were performed to examine the molecular interactions and energetics in a modular capsomere of a murine polyomavirus (MPV) VLP designed to protect against influenza. Insertion of an influenza antigenic module is found to lower the binding energy within the capsomere, and a more active state is observed in Assembly Buffer as compared with that in Stabilization Buffer, which has been experimentally validated through measurements using differential scanning calorimetry. Further in-depth analysis based on free-energy decomposition indicates that destabilized binding can be attributed to electrostatic interaction induced by the chosen antigen module. These results provide molecular insights into the conformational stability of capsomeres and their abilities to be exploited for antigen presentation, and are expected to be beneficial for the biomolecular engineering of VLP vaccines.     

Insight the C-Site Pocket Conformational Changes Responsible for Sirtuin 2 Activity Using Molecular Dynamics Simulations

Sirtuin belongs to a family of typical histone deacetylase which regulates the fundamental cellular biological processes including gene expression, genome stability, mitosis, nutrient metabolism, aging, mitochondrial function, and cell motility. Michael et. al. reported that B-site mutation (Q167A and H187A) decreased the SIRT2 activity but still the structural changes were not reported. Hence, we performed 5 ns molecular dynamics (MD) simulation on SIRT2 Apo-form and complexes with substrate/NAD⁺ and inhibitor of wild type (WT), Q167A, and H187A. The results revealed that the assembly and disassembly of C-site induced by presence of substrate/NAD⁺ and inhibitor, respectively. This assembly and disassembly was mainly due to the interaction between the substrate/NAD⁺ and inhibitor and F96 and the distance between F96 and H187 which are present at the neck of the C-site. MD simulations suggest that the conformational change of L3 plays a major role in assembly and disassembly of C-site. Our current results strongly suggest that the distinct conformational change of L3 as well as the assembly and disassembly of C-site plays an important role in SIRT2 deacetylation function. Our study unveiled the structural changes of SIRT2 in presence of NAD⁺ and inhibitor which should be helpful to improve the inhibitory potency of SIRT2.     

Structural Dynamics of Human Telomeric G-Quadruplex Loops Studied by Molecular Dynamics Simulations

Loops which are linkers connecting G-strands and supporting the G-tetrad core in G-quadruplex are important for biological roles of G-quadruplexes. TTA loop is a common sequence which mainly resides in human telomeric DNA (hTel) G-quadruplex. A series of molecular dynamics (MD) simulations were carried out to investigate the structural dynamics of TTA loops. We found that (1) the TA base pair formed in TTA loops are very stable, the occupied of all hydrogen bonds are more than 0.95. (2) The TA base pair makes the adjacent G-quartet more stable than others. (3) For the edgewise loop and the diagonal loop, most loop bases are stacking with others, only few bases have considerable freedom. (4) The stabilities of these stacking structures are distinct. Part of the loops, especially TA base pairs, and bases stacking with the G-quartet, maintain certain stable conformations in the simulation, but other parts, like TT and TA stacking structures, are not stable enough. For the first time, spontaneous conformational switches of TTA edgewise loops were observed in our long time MD simulations. (5) For double chain reversal loop, it is really hard to maintain a stable conformation in the long time simulation under present force fields (parm99 and parmbsc0), as it has multiple conformations with similar free energies.     

Speed of Conformational Change: Comparing Explicit and Implicit Solvent Molecular Dynamics Simulations

Adequate sampling of conformation space remains challenging in atomistic simulations, especially if the solvent is treated explicitly. Implicit-solvent simulations can speed up conformational sampling significantly. We compare the speed of conformational sampling between two commonly used methods of each class: the explicit-solvent particle mesh Ewald (PME) with TIP3P water model and a popular generalized Born (GB) implicit-solvent model, as implemented in the AMBER package. We systematically investigate small (dihedral angle flips in a protein), large (nucleosome tail collapse and DNA unwrapping), and mixed (folding of a miniprotein) conformational changes, with nominal simulation times ranging from nanoseconds to microseconds depending on system size. The speedups in conformational sampling for GB relative to PME simulations, are highly system- and problem-dependent. Where the simulation temperatures for PME and GB are the same, the corresponding speedups are approximately onefold (small conformational changes), between ∼1- and ∼100-fold (large changes), and approximately sevenfold (mixed case). The effects of temperature on speedup and free-energy landscapes, which may differ substantially between the solvent models, are discussed in detail for the case of miniprotein folding. In addition to speeding up conformational sampling, due to algorithmic differences, the implicit solvent model can be computationally faster for small systems or slower for large systems, depending on the number of solute and solvent atoms. For the conformational changes considered here, the combined speedups are approximately twofold, ∼1- to 60-fold, and ∼50-fold, respectively, in the low solvent viscosity regime afforded by the implicit solvent. For all the systems studied, 1) conformational sampling speedup increases as Langevin collision frequency (effective viscosity) decreases; and 2) conformational sampling speedup is mainly due to reduction in solvent viscosity rather than possible differences in free-energy landscapes between the solvent models.     

A Molecular Dynamics Investigation of Chain Conformational Changes In Compressed Bilayers

Abstract

The conformations of the chains constituting the hydrophilic component of alkyl monolayers and bilayers are investigated by performing molecular dynamics atomistic simulations on these systems at different temperatures. Several monitoring techniques are used to reveal the chain conformations, including atom pair radial distribution functions, evolutions of the torsional angles over thousands of timesteps, frequency distributions of the torsionl angles and ‘snapshot’ plots of the atomic configurations. These methods consistently testify to the stability of the trans (fully extended) character of the strain-free alkyl chains up to room temperature. The chains retain much of this conformation even when the layers are compressed by the application of pressure, to which the chains respond by ‘folding’ at the ends attaching them to the substrate planes while maintaining directions which are mainly normal to these planes. A non-zero gap between the layers is also maintained. A pressure of about 50 kbar abruptly causes all motion in the chains to cease, resulting in a highly ordered lattice structure.     

The Influence of 150-Cavity Binders on the Dynamics of Influenza A Neuraminidases as Revealed by Molecular Dynamics Simulations and Combined Clustering

Neuraminidase inhibitors are the main pharmaceutical agents employed for treatments of influenza infections. The neuraminidase structures typically exhibit a 150-cavity, an exposed pocket that is adjacent to the catalytic site. This site offers promising additional contact points for improving potency of existing pharmaceuticals, as well as generating entirely new candidate inhibitors. Several inhibitors based on known compounds and designed to interact with 150-cavity residues have been reported. However, the dynamics of any of these inhibitors remains unstudied and their viability remains unknown. This work reports the outcome of long-term, all-atom molecular dynamics simulations of four such inhibitors, along with three standard inhibitors for comparison. Each is studied in complex with four representative neuraminidase structures, which are also simulated in the absence of ligands for comparison, resulting in a total simulation time of 9.6µs. Our results demonstrate that standard inhibitors characteristically reduce the mobility of these dynamic proteins, while the 150-binders do not, instead giving rise to many unique conformations. We further describe an improved RMSD-based clustering technique that isolates these conformations – the structures of which are provided to facilitate future molecular docking studies – and reveals their interdependence. We find that this approach confers many advantages over previously described techniques, and the implications for rational drug design are discussed.     

Designing Molecular Dynamics Simulations to Shift Populations of the Conformational States of Calmodulin

We elucidate the mechanisms that lead to population shifts in the conformational states of calcium-loaded calmodulin (Ca²⁺-CaM). We design extensive molecular dynamics simulations to classify the effects that are responsible for adopting occupied conformations available in the ensemble of NMR structures. Electrostatic interactions amongst the different regions of the protein and with its vicinal water are herein mediated by lowering the ionic strength or the pH. Amino acid E31, which is one of the few charged residues whose ionization state is highly sensitive to pH differences in the physiological range, proves to be distinctive in its control of population shifts. E31A mutation at low ionic strength results in a distinct change from an extended to a compact Ca²⁺-CaM conformation within tens of nanoseconds, that otherwise occur on the time scales of microseconds. The kinked linker found in this particular compact form is observed in many of the target-bound forms of Ca²⁺-CaM, increasing the binding affinity. This mutation is unique in controlling C-lobe dynamics by affecting the fluctuations between the EF-hand motif helices. We also monitor the effect of the ionic strength on the conformational multiplicity of Ca²⁺-CaM. By lowering the ionic strength, the tendency of nonspecific anions in water to accumulate near the protein surface increases, especially in the vicinity of the linker. The change in the distribution of ions in the vicinal layer of water allows N- and C- lobes to span a wide variety of relative orientations that are otherwise not observed at physiological ionic strength. E31 protonation restores the conformations associated with physiological environmental conditions even at low ionic strength.     

Hierarchical Conformational Analysis of Native Lysozyme Based on Sub-Millisecond Molecular Dynamics Simulations

Hierarchical organization of free energy landscape (FEL) for native globular proteins has been widely accepted by the biophysics community. However, FEL of native proteins is usually projected onto one or a few dimensions. Here we generated collectively 0.2 milli-second molecular dynamics simulation trajectories in explicit solvent for hen egg white lysozyme (HEWL), and carried out detailed conformational analysis based on backbone torsional degrees of freedom (DOF). Our results demonstrated that at micro-second and coarser temporal resolutions, FEL of HEWL exhibits hub-like topology with crystal structures occupying the dominant structural ensemble that serves as the hub of conformational transitions. However, at 100ns and finer temporal resolutions, conformational substates of HEWL exhibit network-like topology, crystal structures are associated with kinetic traps that are important but not dominant ensembles. Backbone torsional state transitions on time scales ranging from nanoseconds to beyond microseconds were found to be associated with various types of molecular interactions. Even at nanoseconds temporal resolution, the number of conformational substates that are of statistical significance is quite limited. These observations suggest that detailed analysis of conformational substates at multiple temporal resolutions is both important and feasible. Transition state ensembles among various conformational substates at microsecond temporal resolution were observed to be considerably disordered. Life times of these transition state ensembles are found to be nearly independent of the time scales of the participating torsional DOFs.     

Conformational Studies of Two Bradykinin Antagonists by Using Two-dimensional NMR Techniques and Molecular Dynamics Simulations

Abstract

The solution conformations of two potent antagonists of bradykinin (Arg¹-Pro²-Pro³-Gly⁴- Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹), [Aca-¹, DArg⁰, Hyp³, Thi⁵, DPhe⁷,(N-Bzl)Gly⁸]BK (1) and [Aaa- ¹, DArg⁰, Hyp³, Thi⁵,(2-DNal)⁷, Thi⁸]BK (2), were studied by using 2D NMR spectroscopy in DMSO-dg and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe⁷-(N-Bzl)Gly⁸peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a β-turn structure for the Hyp³-Gly⁴ residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe⁷ and (N- Bzl)Gly⁸ in analogue 1 suggests type VI β-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb β-turn comprising residues Ser-Arg⁹ and the βI or βII-turn involving the Pro²-Thi⁵ fragment, whereas peptide 2 shows the tendency towards the formation of type I β-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg¹ and the carboxyl group of Arg⁹. Moreover, the side chain of DArg⁰ is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

Structural Analysis of Human Lysozyme Using Molecular Dynamics Simulations

Abstract

In this study, various molecular dynamics simulations were conducted to investigate the effects of ethanol and temperature on the conformational changes of human lysozyme, which may lead insights into amyloidosis. The analyses of some important structural characteristics, such as backbone root-mean-square deviation, secondary structural stability, radius of gyration, accessible surface area, and hydrophobic contact of the hydrophobic core all show that ethanol tends to destabilize human lysozyme at high temperatures. It can be attributed to that higher temperatures result in the destruction of the native structure of this protein, leading to the exposure of the interior hydrophobic core. At this stage, ethanol plays a role to destroy this region by forming hydrophobic interactions between protein and solvent due to its lower polarity comparing to water. Such newly formed intermolecular interactions accelerate the unfolding of this protein, starting from the core between the a- and β-domains. Our results are in good agreement with the previous hypothesis suggesting that the distortion of the hydrophobic core at the α- and β-interface putatively results in the formation of the initial “seed” for amyloid fibril. Although the present results cannot directly be linked to fibril formation, they still provide valuable insights into amyloidosis of human lysozyme.     

Evolution of Stronger SARS-CoV-2 Variants as Revealed Through the Lens of Molecular Dynamics Simulations

The Protein Journal - Using molecular dynamics simulations, the protein–protein interactions of the receptor-binding domain of the wild-type and seven variants of the severe acute respiratory...     

Conformational Dynamics of Subtilisin-Chymotrypsin Inhibitor 2 Complex by Coarse-Grained Simulations

Abstract

An off-lattice dynamic Monte Carlo (MC) method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2) and subtilisin in both free and complex forms over two time windows, referring to short and long time scales. The conformational dynamics of backbone bonds analysed from several independent trajectories reveal that: Both the inhibitor and the enzyme are restricted in their bond rotations, excluding a few bonds, upon binding; the effect being greatest for the loop regions, and for the inhibitor. A cooperativity in the near-neighbor bond rotations are observed on both time scales, whereas the cooperative rotations of the bonds far along the sequence appear only in the long time window, and the latter time window is where most of the interactions between the inhibitor and the enzyme are observed. Upon binding, the cooperatively rotating parts of the inhibitor and the enzyme are readjusted compared to their free forms, and new correlations appear. The binding loop, although it is the closest contact region, is not the only part of the inhibitor involved in the interactions with the enzyme. Loops 3 and 8 and the helices F and G in bound enzyme and the binding loop of the inhibitor contribute at the most to the collective motions of whole structure on the slow time scale and are apparently important for enzyme-inhibitor interactions and function. The results in general provide evidence for the contribution of the loops with cooperative motions to the extensive communication network of the complex.     

The Dynamic Conformational Cycle of the Group I Chaperonin C-Termini Revealed via Molecular Dynamics Simulation

Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins.