Asymmetric structural motions of the homomeric alpha7 nicotinic receptor ligand binding domain revealed by molecular dynamics simulation

A homology model of the ligand binding domain of the alpha7 nicotinic receptor is constructed based on the acetylcholine-binding protein crystal structure. This structure is refined in a 10 ns molecular dynamics simulation. The modeled structure proves fairly resilient, with no significant changes at the secondary or tertiary structural levels. The hypothesis that the acetylcholine-binding protein template is in the activated or desensitized state, and the absence of a bound agonist in the simulation suggests that the structure may also be relaxing from this state to the activatable state. Candidate motions that take place involve not only the side chains of residues lining the binding sites, but also the subunit positions that determine the overall shape of the receptor. In particular, two nonadjacent subunits move outward, whereas their partners counterclockwise to them move inward, leading to a marginally wider interface between themselves and an overall asymmetric structure. This in turn affects the binding sites, producing two that are more open and characterized by distinct side-chain conformations of W54 and L118, although motions of the side chains of all residues in every binding site still contribute to a reduction in binding site size, especially the outward motion of W148, which hinders acetylcholine binding. The Cys loop at the membrane interface also displays some flexibility. Although the short simulation timescale is unlikely to sample adequately all the conformational states, the pattern of observed motions suggests how ligand binding may correlate with larger-scale subunit motions that would connect with the transmembrane region that controls the passage of ions. Furthermore, the shape of the asymmetry with binding sites of differing affinity for acetylcholine, characteristic of other nicotinic receptors, may be a natural property of the relaxed, activatable state of alpha7.     

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50–60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm² in the search for a second endosome with which to interact.     

A noncanonical binding site of chloramphenicol revealed via molecular dynamics simulations

Chloramphenicol, an antibiotic belonging to the family of amphenicols, is an inhibitor of translation. On the basis of X–ray structural analysis of the binding of chloramphenicol to free bacterial ribosomes, the chloramphenicol action mechanism that consists in preventing the binding of aminoacyl-tRNA to the A–site of the large subunit of the ribosome was adopted. However, the known structures of chloramphenicol complexes with bacterial ribosomes poorly explain the results of the experiments on the chemical modification of 23S rRNA, the resistance to chloramphenicol caused by mutations in 23S rRNA and, which is particularly important, the selectivity of chloramphenicol in suppression of translation, depending on the amino acid sequence of the nascent peptide. In the present study the putative structure of the chloramphenicol complex with a bacterial ribosome in the A,A/P,P–state has been obtained by molecular dynamics simulations methods. The proposed structure of the complex allows us to explain the results of biochemical studies of the interaction of chloramphenicol with the bacterial ribosome.     

Structural dynamics of precursor and product of the RNA enzyme from the hepatitis delta virus as revealed by molecular dynamics simulations

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme involved in the replication of a human pathogen, the hepatitis delta virus. Recent crystal structures of the precursor and product of self-cleavage, together with detailed kinetic analyses, have led to hypotheses on the catalytic strategies employed by the HDV ribozyme. We report molecular dynamics (MD) simulations (approximately 120 ns total simulation time) to test the plausibility that specific conformational rearrangements are involved in catalysis. Site-specific self-cleavage requires cytidine in position 75 (C75). A precursor simulation with unprotonated C75 reveals a rather weak dynamic binding of C75 in the catalytic pocket with spontaneous, transient formation of a H-bond between U-1(O2') and C75(N3). This H-bond would be required for C75 to act as the general base. Upon protonation in the precursor, C75H+ has a tendency to move towards its product location and establish a firm H-bonding network within the catalytic pocket. However, a C75H+(N3)-G1(O5') H-bond, which would be expected if C75 acted as a general acid catalyst, is not observed on the present simulation timescale. The adjacent loop L3 is relatively dynamic and may serve as a flexible structural element, possibly gated by the closing U20.G25 base-pair, to facilitate a conformational switch induced by a protonated C75H+. L3 also controls the electrostatic environment of the catalytic core, which in turn may modulate C75 base strength and metal ion binding. We find that a distant RNA tertiary interaction involving a protonated cytidine (C41) becomes unstable when left unprotonated, leading to disruptive conformational rearrangements adjacent to the catalytic core. A Na ion temporarily compensates for the loss of the protonated hydrogen bond, which is strikingly consistent with the experimentally observed synergy between low pH and high Na+ concentrations in mediating residual self-cleavage of the HDV ribozyme in the absence of divalents.     

Assembly of lipoprotein particles revealed by coarse-grained molecular dynamics simulations

High-density lipoproteins (HDL) function as cholesterol transporters, facilitating the removal of excess cholesterol from the body. Due to the heterogeneity of native HDL particles (both in size and shape), the details on how these protein-lipid particles form and the structure they assume in their lipid-associated states are not well characterized. We report here a study of the self-assembly of discoidal HDL particles using coarse-grained (CG) molecular dynamics. The microsecond simulations reveal the self-assembly of HDL particles from disordered protein-lipid complexes to form structures containing many of the features of the generally accepted double-belt model for discoidal HDL particles. HDL assembly is found to proceed in two broad steps, aggregation of proteins and lipids driven by the hydrophobic effect which occurs on a approximately 1 micros time scale, followed by the optimization of the protein structure driven by increasingly specific protein-protein interactions.     

Thermodynamic coupling between activation and inactivation gating in potassium channels revealed by free energy molecular dynamics simulations

The amount of ionic current flowing through K(+) channels is determined by the interplay between two separate time-dependent processes: activation and inactivation gating. Activation is concerned with the stimulus-dependent opening of the main intracellular gate, whereas inactivation is a spontaneous conformational transition of the selectivity filter toward a nonconductive state occurring on a variety of timescales. A recent analysis of multiple x-ray structures of open and partially open KcsA channels revealed the mechanism by which movements of the inner activation gate, formed by the inner helices from the four subunits of the pore domain, bias the conformational changes at the selectivity filter toward a nonconductive inactivated state. This analysis highlighted the important role of Phe103, a residue located along the inner helix, near the hinge position associated with the opening of the intracellular gate. In the present study, we use free energy perturbation molecular dynamics simulations (FEP/MD) to quantitatively elucidate the thermodynamic basis for the coupling between the intracellular gate and the selectivity filter. The results of the FEP/MD calculations are in good agreement with experiments, and further analysis of the repulsive, van der Waals dispersive, and electrostatic free energy contributions reveals that the energetic basis underlying the absence of inactivation in the F103A mutation in KcsA is the absence of the unfavorable steric interaction occurring with the large Ile100 side chain in a neighboring subunit when the intracellular gate is open and the selectivity filter is in a conductive conformation. Macroscopic current analysis shows that the I100A mutant indeed relieves inactivation in KcsA, but to a lesser extent than the F103A mutant.     

Dissolution of cellulose in ionic liquid and water mixtures as revealed by molecular dynamics simulations

Abstract

Increasing population growth and industrialization are continuously oppressing the existing energy resources, elevating the pollution and global fuel demand. Various alternate energy resources can be utilized to cope with these problems in an environment-friendly fashion. Currently, bioethanol (sugarcane, corn-derived) is one of the most widely consumed biofuels in the world. Lignocellulosic biomass is yet another attractive resource for sustainable bioethanol production. Pretreatment step plays a crucial role in the lignocellulose to bioethanol conversion by enhancing cellulose susceptibility to enzymatic hydrolysis. However, economical lignocellulose pretreatment still remains a challenging job. Ionic liquids (ILs), especially 1-ethyl-3-methylimidazolium acetate (EmimAc), is an efficient solvent for cellulose dissolution with improved enzymatic saccharification kinetics. To increase the process efficiency as well as recyclability of IL, water is shown as a compatible cosolvent for lignocellulosic pretreatment. The performance analysis of IL–water mixture based on the molecular level understanding may help to design effective pretreatment solvents. In this study, all-atom molecular dynamics simulation has been performed using EmimAc–water mixtures to understand the behavior of cellulose microcrystal containing eight glucose octamers at room and pretreatment temperatures. High-temperature simulation results show effective cellulose chain separation where cellulose–acetate interaction is found to be the driving force behind dissolution. It is also observed that pretreatment with 50 and 80% IL mixture is efficient in decreasing cellulose crystallinity. At a high IL concentration, water exists in a clustered network which gradually spans into the medium with increasing water fraction leading to loss of its cosolvation activity.

Communicated by Ramaswamy H. Sarma     

Mechanisms for allosteric activation of protease DegS by ligand binding and oligomerization as revealed from molecular dynamics simulations

A local perturbation of a protein may lead to functional changes at some distal site, a phenomenon denoted as allostery. Here, we study the allosteric control of a protease using molecular dynamics simulations. The system considered is the bacterial protein DegS which includes a protease domain activated on ligand binding to an adjacent PDZ domain. Starting from crystallographic structures of DegS homo‐trimers, we perform simulations of the ligand‐free and ‐bound state of DegS at equilibrium. Considering a single protomer only, the trimeric state was mimicked by applying restraints on the residues in contact with other protomers in the DegS trimer. In addition, the bound state was also simulated without any restraints to mimic the monomer. Our results suggest that not only ligand release but also disassembly of a DegS trimer inhibits proteolytic activity. Considering various observables for structural changes, we infer allosteric pathways from the interface with other protomers to the active site. Moreover, we study how ligand release leads to (i) catalytically relevant changes involving residues 199–201 and (ii) a transition from a stretched to a bent conformation for residues 217–219 (which prohibits proper substrate binding). Finally, based on ligand‐induced C_α shifts we identify residues in contact with other protomers in the DegS trimer that likely transduce the perturbation from ligand release from a given protomer to adjacent protomers. These residues likely play a key role in the experimentally known effect of ligand release from a protomer on the proteolytic activity of the other protomers. Proteins 2016; 84:1690–1705. © 2016 Wiley Periodicals, Inc.     

Inhibitory mechanism of caspase-6 phosphorylation revealed by crystal structures, molecular dynamics simulations, and biochemical assays

The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser(257) with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser(257) inhibited self-activation of CASP6 zymogen by "locking" the enzyme in the TEVD(193)-bound "inhibited state." The structural and biochemical results also showed that phosphorylation on Ser(257) inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.     

Conformational dynamics of the ligand-binding domain of inward rectifier K channels as revealed by molecular dynamics simulations: toward an understanding of Kir channel gating

Inward rectifier (Kir) potassium channels are characterized by two transmembrane helices per subunit, plus an intracellular C-terminal domain that controls channel gating in response to changes in concentration of various ligands. Based on the crystal structure of the tetrameric C-terminal domain of Kir3.1, it is possible to build a homology model of the ATP-binding C-terminal domain of Kir6.2. Molecular dynamics simulations have been used to probe the dynamics of Kir C-terminal domains and to explore the relationship between their dynamics and possible mechanisms of channel gating. Multiple simulations, each of 10 ns duration, have been performed for Kir3.1 (crystal structure) and Kir6.2 (homology model), in both their monomeric and tetrameric forms. The Kir6.2 simulations were performed with and without bound ATP. The results of the simulations reveal comparable conformational stability for the crystal structure and the homology model. There is some decrease in conformational flexibility when comparing the monomers with the tetramers, corresponding mainly to the subunit interfaces in the tetramer. The beta-phosphate of ATP interacts with the side chain of K185 in the Kir6.2 model and simulations. The flexibility of the Kir6.2 tetramer is not changed greatly by the presence of bound ATP, other than in two loop regions. Principal components analysis of the simulated dynamics suggests loss of symmetry in both the Kir3.1 and Kir6.2 tetramers, consistent with "dimer-of-dimers" motion of subunits in C-terminal domains of the corresponding Kir channels. This is suggestive of a gating model in which a transition between exact tetrameric symmetry and dimer-of-dimers symmetry is associated with a change in transmembrane helix packing coupled to gating of the channel. Dimer-of-dimers motion of the C-terminal domain tetramer is also supported by coarse-grained (anisotropic network model) calculations. It is of interest that loss of exact rotational symmetry has also been suggested to play a role in gating in the bacterial Kir homolog, KirBac1.1, and in the nicotinic acetylcholine receptor channel.     

Factors influencing local membrane curvature induction by N-BAR domains as revealed by molecular dynamics simulations

N-BAR domains are protein modules that bind to and induce curvature in membranes via a charged concave surface and N-terminal amphipathic helices. Recently, molecular dynamics simulations have demonstrated that the N-BAR domain can induce a strong local curvature that matches the curvature of the BAR domain surface facing the bilayer. Here we present further molecular dynamics simulations that examine in greater detail the roles of the concave surface and amphipathic helices in driving local membrane curvature. We find that the strong curvature induction observed in our previous simulations requires the stable presentation of the charged concave surface to the membrane and is not driven by the membrane-embedded amphipathic helices. Nevertheless, without these amphipathic helices embedded in the membrane, the N-BAR domain does not maintain a close association with the bilayer, and fails to drive membrane curvature. Increasing the membrane negative charge through the addition of PIP₂ facilitates closer association with the membrane in the absence of embedded helices. At sufficiently high concentrations, amphipathic helices embedded in the membrane drive membrane curvature independently of the BAR domain.     

Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations

The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation. RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA. MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale. We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered. At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively. These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.     

Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei

RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.     

Structure and interactions of RecA: plasticity revealed by molecular dynamics simulations

Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from Mycobacterium tuberculosis, Mycobacterium smegmatis and Escherichia coli and their Adenosine triphosphate (ATP) complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural framework within which RecA molecules from different eubacteria function. Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.     

Conformational states of the glucocorticoid receptor DNA-binding domain from molecular dynamics simulations

Molecular dynamics simulations (MD) have been performed on variant crystal and NMR-derived structures of the glucocorticoid receptor DNA-binding domain (GR DBD). A loop region five residues long, the so-called D-box, exhibits significant flexibility, and transient perturbations of the tetrahedral geometry of two structurally important Cys4 zinc finger are seen, coupled to conformational changes in the D-box. In some cases, one of the Cys ligands to zinc exchanges with water, although no global distortion of the protein structure is observed. Thus, from MD simulation, dynamics of the D-box could partly be explained by solvent effects in conjunction with structural reformation of the zinc finger.     

Insights into correlated motions and long-range interactions in CheY derived from molecular dynamics simulations

CheY is a response regulator protein involved in bacterial chemotaxis. Much is known about its active and inactive conformations, but little is known about the mechanisms underlying long-range interactions or correlated motions. To investigate these events, molecular dynamics simulations were performed on the unphosphorylated, inactive structure from Salmonella typhimurium and the CheY-BeF(3)(-) active mimic structure (with BeF(3)(-) removed) from Escherichia coli. Simulations utilized both sequences in each conformation to discriminate sequence- and structure-specific behavior. The previously identified conformational differences between the inactive and active conformations of the strand-4-helix-4 loop, which are present in these simulations, arise from the structural, and not the sequence, differences. The simulations identify previously unreported structure-specific flexibility features in this loop and sequence-specific flexibility features in other regions of the protein. Both structure- and sequence-specific long-range interactions are observed in the active and inactive ensembles. In the inactive ensemble, two distinct mechanisms based on Thr-87 or Ile-95 rotameric forms, are observed for the previously identified g+ and g- rotamer sampling by Tyr-106. These molecular dynamics simulations have thus identified both sequence- and structure-specific differences in flexibility, long-range interactions, and rotameric form of key residues. Potential biological consequences of differential flexibility and long-range correlated motion are discussed.