Structural implication for the impaired binding of W150A mutant LOX-1 to oxidized low density lipoprotein, OxLDL

Lectin-like oxidized lipoprotein (OxLDL) receptor 1, LOX-1, is the major OxLDL receptor expressed on vascular endothelial cells. We have previously reported the ligand-recognition mode of LOX-1 based on the crystal structure of the ligand binding domain (C-type lectin-like domain, CTLD) and surface plasmon resonance analysis, which suggested that the functional significance of the CTLD dimer (the 'canonical' dimer) is to harbor the characteristic "basic spine" on its surface. In this study, we have identified the key inter-domain interactions in retaining the canonical CTLD dimer by X-ray structural analysis of the inactive mutant W150A CTLD. The canonical CTLD dimer forms through tight hydrophobic interactions, in which W150 engages in a lock-and-key manner and represents the main interaction. The loss of the Trp ring by mutation to Ala prevents the formation of the canonical dimer, as elucidated from docking calculations using the crystal structure of W150A CTLD. The results emphasize that the canonically formed CTLD dimer is essential for LOX-1 to bind to OxLDL, which supports our proposed view that the basic spine surface present in the correctly formed dimer plays a primal role in OxLDL recognition. This concept provides insight into the pathogenic pattern recognized by LOX-1 as a member of the pattern recognition receptors.     

Molecular dynamics simulation as a tool for assessment of drug binding property of human serum albumin

Human serum albumin (HSA) is a major plasma protein and binding of drugs with this plasma protein has a great importance. It possess esterase activity which can cleave the drugs containing ester bond and thus, can regulate the effect of drugs. Till date no systematic study has been done to analyse binding of such drugs and to compare the results with the drugs which do not have ester bond. Therefore, in the present study two different categories—ester and non-ester drugs have been considered to analyse their interaction with HSA at two principle drug binding sites using molecular modelling tools. It is observed that the drugs irrespective of ester or non-ester nature prefer either Sudlow site I or II by hydrogen bond and hydrophobic interactions. The information obtained from the study can assist to study pharmacokinetics of the drugs and that in turn will help in noval drug discoveries.     

Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament

Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the “Driven by Detachment (DbD)” mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.     

Molecular dynamics simulation and density functional theory studies on the active pocket for the binding of paclitaxel to tubulin

Paclitaxel (PTX) is used to treat various cancers, but it also causes serious side effects and resistance. To better design similar compounds with less toxicity and more activity against drug-resistant tumors, it is important to clearly understand the PTX-binding pocket formed by the key residues of active sites on β-tubulin. Using a docking method, molecular dynamics (MD) simulation and density functional theory (DFT), we identified some residues (such as Arg278, Asp26, Asp226, Glu22, Glu27, His229, Arg369, Lys218, Ser277 and Thr276) on β-tubulin that are the active sites responsible for interaction with PTX. Another two residues, Leu371 and Gly279, also likely serve as active sites. Most of these sites contact with the “southern hemisphere” of PTX; only one key residue interacts with the “northern hemisphere” of PTX. These key residues can be divided into four groups, which serve as active compositions in the formation of an active pocket for PTX binding to β-tubulin. This active binding pocket enables a very strong interaction (the strength is predicted to be in the range of −327.8 to −365.7 kJ mol⁻¹) between β-tubulin and PTX, with various orientated conformations. This strong interaction means that PTX possesses a high level of activity against cancer cells, a result that is in good agreement with the clinical mechanism of PTX. The described PTX pocket and key active residues will be applied to probe the mechanism of tumor cells resistant to PTX, and to design novel analogs with superior properties.     

Molecular dynamics simulation of the docking of substrates to proteins

A simple method is described to perform docking of subtrates to proteins or probes to receptor molecules by a modification of molecular dynamics simulations. The method consists of a separation of the center-of-mass motion of the substrate from its internal and rotational motions, and a separate coupling to different thermal baths for both types of motion of the substrate and for the motion of the receptor. Thus the temperatures and the time constants of coupling to the baths can be arbitrarily varied for these three types of motion, allowing either a frozen or a flexible receptor and allowing control of search rate without disturbance of internal structure. In addition, an extra repulsive term between substrate and protein was applied to smooth the interaction. The method was applied to a model substrate docking onto a model surface, and to the docking of phosphocholine onto immunoglobulin McPC603, in both cases with a frozen receptor. Using transrational temperatures of the substrate in the range of 1300–1700 K and room temperature for the internal degrees of freedom of the substrate, an efficient nontrapping exploratory search (“helicopter view”) is obtained, which visits the correct binding sites. Low energy conformations can then be further investigated by separate search or by dynamic simulated annealing. In both cases the correct minima were identified. The possibility to work with flexible receptors is discussed. © 1994 Wiley-Liss, Inc.     

Molecular dynamics simulation study of AG10 and tafamidis binding to the Val122Ile transthyretin variant

Molecular dynamics (MD) simulations were used to investigate the binding of four ligands to the Val122Ile mutant of the protein transthyretin. Dissociation, misfolding, and subsequent aggregation of mutated transthyretin proteins are associated with the disease Familial Amyloidal Cardiomyopathy. The ligands investigated were the drug candidate AG10 and its decarboxy and N-methyl derivatives along with the drug tafamidis. These ligands bound to the receptor in two halogen binding pockets (HBP) designated AB and A’B’. Inter-ligand distances, solvent accessible surface areas, root mean squared deviation measurements, and extracted structures showed very little change in the AG10 ligands' conformations or locations within the HBP during the MD simulation. In addition, the AG10 ligands experienced stable, two-point interactions with the protein by forming hydrogen bonds with Ser-117 residues in both the AB and A’B’ binding pockets and Lysine-15 residues found near the surface of the receptor. Distance measurements showed these H-bonds formed simultaneously during the MD simulation. Removal of the AG10 carboxylate functional group to form decarboxy-AG10 disrupted this two-point interaction causing the ligand in the AB pocket to undergo a conformational change during the MD simulation. Likewise, addition of a methyl group to the AG10 hydrazone functional group also disrupted the two-point interaction by decreasing hydrogen bonding interactions with the receptor. Finally, MD simulations showed that the tafamidis ligands experienced fewer hydrogen bonding interactions than AG10 with the protein receptor. The tafamidis ligand in pocket A’B’ was also found to move deeper into the HBP during the MD simulation.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.     

Molecular dynamics investigation of an antibody binding site by the fluorescence-photochrome method

A combined fluorescence-photochrome approach was used for investigation of the molecular dynamics antiDNP antibody binding site and its cavity. A 4-(N-2,4-dinitrophenylamino)-4'-(N,N'-dimethylamino)stilbene (StDNP) fluorescence DNP analog was incorporated into the antibody binding site. This was followed by measurements of fluorescence and photochrome parameters such as the StDNP excitation and emission spectra, fluorescence lifetime, steady-state and time-resolved fluorescence polarization, kinetics of trans-cis and cis-trans photoisomerization, and fluorescence quenching by nitroxide radicals freely diffused in solution. In parallel, computational modeling studies on the location and dynamics of DNP/TEMPO spin-label (NslDNP) and StDNP guests within a model of the binding site were performed. When all the experimental evidence is considered (including data from the antibody X-ray study), one can conclude that wobbling of the Trp 91 L/Trp 96 H binding-site.bound-hapten moiety (StDNP), can be responsible for the label's nanosecond dynamics monitored by fluorescence polarization techniques. A similar conclusion may be reached as a result of data analysis on NslDNP mobility within the antibody binding site. The mobility of Trp 91 L and Trp 96 H moieties provides the induced fit needed for effective stacking and release of the DNP epitope. Analysis of the above-mentioned data allows one to explore the mechanism of the probe's movement within the binding site and enables one to discuss the local dynamics of the binding site region. The combined fluorescence-photochrome approach can be used for investigation of local medium molecular dynamics in the immediate vicinity of specific sites of proteins and nucleic acids, as well as for other biologically important structures and synthetic analogues.     

Molecular dynamics simulation of the membrane binding and disruption mechanisms by antimicrobial scorpion venom-derived peptides

Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from venom of the African scorpion Pandinus imperator with high antimicrobial activity against Gram-positive bacteria and less active against Gram-negative bacteria, however it has demonstrated strong hemolytic activity against sheep red blood cells. In the chemically synthesized Pin2GVG analog, the GVG motif grants it low hemolytic activity while keeping its antimicrobial activity. In this work, we performed 12 μs all-atom molecular dynamics simulation of the antimicrobial peptides (AMPs) Pin2 and Pin2GVG to explore their adsorption mechanism and the role of their constituent amino acid residues when interacting with pure POPC and pure POPG membrane bilayers. Starting from an α-helical conformation, both AMPs are attracted at different rates to the POPC and POPG bilayer surfaces due to the electrostatic interaction between the positively charged amino acid residues and the charged moieties of the membranes. Since POPG is an anionic membrane, the PAMs adhesion is stronger to the POPG membrane than to the POPC membrane and they are stabilized more rapidly. This study reveals that, before the insertion begins, Pin2 and Pin2GVG remained partially folded in the POPC surface during the first 300 and 600 ns, respectively, while they are mostly unfolded in the POPG surface during most of the simulation time. The unfolded structures provide for a large number of intermolecular hydrogen bonds and stronger electrostatic interactions with the POPG surface. The results show that the aromatic residues at the N-terminus of Pin2 initiate the insertion process in both POPC and POPG bilayers. As for Pin2GVG in POPC the C-terminus residues seem to initiate the insertion process while in POPG this process seems to be slowed down due to a strong electrostatic attraction. The membrane conformational effects upon PAMs binding are measured in terms of the area per lipid and the contact surface area. Several replicas of the systems lead to the same observations.     

Molecular dynamics study of the binding of phenylalanine stereoisomers to thermolysin

The stereospecificity in binding of phenylalanine as inhibitor in the active site of the thermolysin, has been investigated by means of molecular dynamics simulations using free energy integration techniques. The difference in the free energy of binding was found to be 2.0 ± 1.8 kJ/mol in favour of the D-form. This agrees with the experimental value, 2.8 kJ/mol. The result was obtained using a standard empirical force field (that of GROMOS). A different force field with 30% bigger charges (more like ab initio charges) was also tried. This resulted in less fluctuations and a more precise binding, but in a free energy. difference that was clearly larger than the experimental one. The phenylalanine backbone is located close to the zinc atom and the ring stays in the hydrophobic pocket in both the cases. The two stereoisomers differ mainly in the orientation of the backbone plane with respect to the active site and the rotational state of the dihedral around the C---CO^β bond.     

Molecular dynamics simulation study on the high-pressure behaviour of an AK16 peptide

While most proteins unfold under high-pressure conditions, some high-pressure experiments suggest that an AK16 peptide forms more helical structures. In order to understand this abnormality, molecular dynamics simulations with the simulated tempering method for the isobaric–isothermal ensemble were performed in a wide pressure range from 0.1 MPa to 1.4 GPa. It was found that the fraction of the folded state decreases once and increases after that with increasing pressure. The partial molar volume change from the folded state to unfolded state increases monotonically from a negative value to a positive value with pressure. The behaviour under high-pressure conditions is consistent with the experimental results. The radius of gyration of highly helical structures decreases with increasing pressure. Moreover, interatomic distances of AK16 become shorter at high pressure than at low pressure. These behaviours indicate that the helical structures are squeezed by high pressure.     

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71–Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71–Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71–Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95–Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α–β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23     

Molecular dynamics simulation of interleukin-2 and its complex and determination of the binding free energy

Interleukin-2 (IL-2) protein belongs to the signal modulator cytokine's family and therefore it is prevalent for immunological responses. It has been identified as a centrally important potential drug target for the inhibition of protein-protein interactions; so as to suppress the immunological responses associated with autoimmune, inflammatory and immunological diseases, and cancer. In the present work, we have performed two independent 100?ns of molecular dynamics (MD) simulations on the apo IL-2 protein and its ligand-bound complex (with a potent inhibitor FRG), to study the effect of inhibitor binding on the dynamics and stability of the protein. The calculation of binding free energy via post-processing end state method of Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) has inferred a good correlation in accordance with the already reported experimental data, demonstrating that the free energy of binding calculated by the two methods has no significant difference. The investigation of individual components of free energy revealed that the association of IL-2 protein with FRG ligand is primarily driven by the van der Waals energy contribution that represents the non-polar/hydrophobic energy contribution as dominant in this case of ligand binding.     

Molecular dynamics simulations and free energy calculations of netropsin and distamycin binding to an AAAAA DNA binding site

Molecular dynamics simulations have been performed on netropsin in two different charge states and on distamycin binding to the minor groove of the DNA duplex d(CGCGAAAAACGCG)·d(CGCGTTTTTCGCG). The relative free energy of binding of the two non-covalently interacting ligands was calculated using the thermodynamic integration method and reflects the experimental result. From 2 ns simulations of the ligands free in solution and when bound to DNA, the mobility and the hydrogen-bonding patterns of the ligands were studied, as well as their hydration. It is shown that even though distamycin is less hydrated than netropsin, the loss of ligand–solvent interactions is very similar for both ligands. The relative mobilities of the ligands in their bound and free forms indicate a larger entropic penalty for distamycin when binding to the minor groove compared with netropsin, partially explaining the lower binding affinity of the distamycin molecule. The detailed structural and energetic insights obtained from the molecular dynamics simulations allow for a better understanding of the factors determining ligand–DNA binding.     

Protamine may have anti-atherogenic potential by inhibiting the binding of oxidized-low density lipoprotein to LOX-1

Oxidized low-density lipoprotein (ox-LDL) leads to atherosclerosis via lectin-like oxidized lipoprotein receptor-1 (LOX-1), one of the major receptor for ox-LDL. Inhibition of the binding of ox-LDL to LOX-1 decreases the proinflammatory and atherosclerotic events. The aim of the present study was to investigate whether protamine, a polybasic nuclear protein, interferes the binding of ox-LDL to LOX-1. Using sandwich ELISA with newly generated antibody, we measured the blocking effect of protamine on the binding of ox-LDL to LOX-1. Protamine dose-dependently inhibited the binding of ox-LDL to LOX-1. DiI-labeled ox-LDL uptake assay in two types of cultured human endothelial cells was performed with fluorescence microplate reader. Activation of extracellular-signal-regulated kinase (ERK)1/2 by ox-LDL was analyzed by immunoblotting. We found that protamine suppressed uptake of ox-LDL in endothelial cells and inhibited ERK1/2 activation by ox-LDL. These results suggest that protamine may possess anti-atherogenic potential by inhibiting ox-LDL binding to LOX-1 through electrostatic interactions.     

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural L-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, L-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing L-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to L and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of L-type prodrugs to assure their efficient metabolic processing.     

Molecular dynamics simulation reveals preorganization of the chloroplast FtsY towards complex formation induced by GTP binding

Two GTPases in the signal recognition particle (SRP) and SRP receptor (SR) interact with one another to mediate the cotranslational protein targeting pathway. Previous studies have shown that a universally conserved SRP RNA facilitates an efficient SRP–SR interaction in the presence of a signal sequence bound to SRP. However, a remarkable exception has been found in chloroplast SRP (cpSRP) pathway, in which the SRP RNA is missing. Based on biochemical and structural analyses, it is proposed that free cpSRP receptor (cpFtsY) has already been preorganized into a closed state for efficient cpSRP–cpFtsY association. However, no direct evidence has been reported to support this postulation thus far. In this study, we characterized the structural dynamics of cpFtsY and its conformational rearrangements induced by GTP binding using molecular dynamics (MD) simulations. Our results showed that the GTP-binding event triggered substantial conformational changes in free cpFtsY, including the relative orientation of N–G domain and several conserved motifs that are critical in complex formation. These rearrangements enabled the cpFtsY to relax into a preorganized ‘closed’ state that favored the formation of a stable complex with cpSRP54. Interestingly, the intrinsic flexibility of αN1 helix facilitated these rearrangements. In addition, GTP binding in cpFtsY was mediated by conserved residues that have been shown in other SRP GTPases. These findings suggested that GTP-bound cpFtsY could fluctuate into conformations that are favorable to form the stable complex, providing explanation of why SRP–SR interaction bypasses the requirement of the SRP RNA at a molecular level.     

Molecular dynamics simulations of human glutathione transferase P1-1: analysis of the induced-fit mechanism by GSH binding.

We report here a 1-ns molecular dynamics simulation on the ligand-free monomer of human glutathione transferase P1-1 in bulk water. The average conformation obtained from the last 500 ps of simulation is taken as a model for the apo-structure of this protein and compared to the available crystallographic data. Remarkable changes in the tertiary structure take place during the simulation and are ascribed to the removal of the ligand. They support an induced fit mechanism occurring upon glutathione binding, whose major features can be described in detail. A portion of helix 2 (residues 42-50), which participates in the formation of the active site, undergoes the most prominent conformational changes. Other protein segments, such as the C-terminal loop and helix 4, also show relevant structural rearrangements. All these transitions cause a significant shielding from the solvent of the hydrophobic binding site of the co-substrate, whose exposed surface goes from 4.6 nm(2) in the holo-structure to about 3.1 nm(2) in the apo-conformation. The results of this simulation are consistent with numerous experimental observations previously obtained on GST P1-1 and provide new insights for their explanation at the molecular level. Proteins 1999;37:1-9.